Capped mRNA

Introduction

Most naturally occurring eukaryotic mRNAs and many viral RNAs have a modified nucleotide structure known as a "cap" on their 5' terminus. In vivo, the cap structure is involved in the initiation of protein synthesis and in mRNA processing and stability. Many viral RNAs are infectious only when capped, and uncapped RNAs introduced into cells via transfection or microinjection are rapidly degraded by cellular RNases. Therefore, RNA prepared for microinjection into oocytes or for transfection into eukaryotic cells should be capped. In vitro, capped mRNAs are translated more efficiently than uncapped transcripts in rabbit reticulocyte lysate or wheat germ translation systems. The trimethylated cap is involved in  RNA transport and RNA translation in nematodes.  Transcripts synthesized for use in in vitro RNA splicing experiments may also need to be capped.

The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 μg RNA per 20 μl reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed. Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability, efficient translation, nuclear transport and splicing. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or  co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications such as generating viral genomic RNA for reverse-genetic systems and crystallographic studies of cap binding proteins such as eIF4E.

The 5' cap structure improves stability of the RNA and translation efficiency, and hence is important for microinjection and transfection experiments. The co-transcriptional capping method generates 40-50 μg of ~80% capped RNA. It is important to note that using 3'-0-Me-m7G(5')ppp(5')G RNA cap analog (ARCA), which is blocked at the 3'-hydroxyl of the m7G ensures incorporation of the cap in the correct orientation. To cap larger quantities of RNA it is possible to scale up the standard reactions for both capping methods. However, using the Vaccinia Capping System would be a more feasible option cost-wise since co-transcriptional capping utilizes cap analog, which is a relatively expensive component. The Vaccinia Capping System is also a better method since it has capping efficiency of almost 100%

NEB T7 mRNA Protocol

Kit: T7 High Yield RNA Synthesis Kit, Cat #: E2040S

Plasmid Templates

• Digest plasmid with an enzyme to generate blunt ends (EcoRV) or 5´-overhangs (e.g., BamH1). Select site should be at the downstream of the insert to be transcribed.
• After linearization, use spin column to purify DNA. Elute with water.

PCR product:

 100–500 ng of PCR fragments can be used in a 20 μl in vitro transcription reaction.

PCR primer design:

F: TAA TAC GAC TCA CTA TAG GG + 21 bp of the 5' end of GOI.

R: Gene specific primer.

Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

Make 20 mM GTP solution by combining 2 μl of 20 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.
Prepare cap analog at the concentration of 40 mM.

Assemble the reaction at room temperature in the following order:

Nuclease-free water	X μl
10X Reaction Buffer	2 μl
ATP (100 mM)	2 μl	10 mM final
UTP (100 mM)	2 μl	10 mM final
CTP (100 mM)	2 μl	10 mM final
GTP (20 mM)	2 μl	2 mM final
Cap Analog (40 mM)	4 μl	8 mM final
Template DNA	X μl	1 μg
T7 RNA Polymerase Mix	2 μl
Total reaction volume	20 μl

Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours.

The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used.

Table 1. Effect of cap analog:GTP ratios on RNA yield

Cap Analog: GTP Ratio	Concentration of Cap Analog: GTP (mM)	RNA Yield (μg) in 2 hours	% Capped RNA
0:1	0:10	180	0
1:1	5:5	90–120	50
2:1	6.7:3.3	60–90	67
4:1	8:2	40–50	80
8:1	8.9:1.1	20–25	89

To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.

Proceed with purification of synthesized RNA using a spin column

PCR Template Preparation
1. Cloning of IDT gene fragment into pMiniT2 or pJet1.2
2683 (DS1)
TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA

2. Make miniprep, send miniprep DNA out for sequencing (Genewiz, Macrogen)
3, PCR  
1 ul of plasmid (50~100 ng)
1 ul of 10 uM T7-mRNA-F3 TAATACGACTCACTATAAGGAGG
1 ul 1 uM 2683-R2 2683-R2 TTACTTGTCGTCATCGTCTTTGTAGTCACCAGCTGTCCAACCTGAAG (CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAA)
1 ul 10 uM Flag-R 
TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG (CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)
21 ul H2O
+ 25 ul 2 X Phusion PCR mix (NEB)

PCR:
2 min at 95 oC
30 cycles of 95 oC 30 sec, 55 oC 30 sec, 72 oC 1 min
7 min at 72 oC.
Final Product 802 bp
TAATACGACTCACTATAAGGAGG
ccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTT
CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA
MRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGDYKDDDDK

purify PCR fragment using Qiagen gel purification kit

Use 1 ug for IVT

5’AG 3’ for the CleanCap AG reagent, N-7113=

https://www.tebu-bio.com/blog/2018/03/13/tips-to-modify-mrna-for-in-vivo-applications-and-delivery-optimisation/

CleanCap reagents are easy to use. They are simply compatible with wild-type T7 polymerase. You apply the same quantity for CleanCap and the rNTP (CTP, UTP, ATP, GTP). It could be 5mM of each depending on the usual protocol. Should use ENB's T7-FlashScribe in vitro transcription kit because of its high yield production (180µg from 1µg)!

CleanCap AG is optimized for production of mRNA that initiates with adenosine as the first base, guanosine as the second base, and guanosine as the third base, so AGG.

Unfortunately, most conventional transcripts start with 5’-GGG.

It would be a T7 promoter sequence like that 5’TAATACGACTCACTATAGGGNNNNNNNNNNNN… 3’.

So for optimal results with the CleanCap AG reagent, you would need have the three bases after the TATA box be AGG.

Thus the best T7 promoter sequence would be 5’TAATACGACTCACTATAAGGNNNNNNNNNNNN… 3’

Using that sequence with the CleanCap AG reagent, N-7113, you will get 95-98% capped mRNA with the natural CAP1 structure.

Keeping the benefit of CAP1 structure If you cannot optimize the T7 promoter sequence, you should use the CleanCap GG reagent, N-7133.

The capping efficiency would be similar to the ARCA (it could be 80%), but you will still benefit of a natural CAP1 structure in place of the CAP0 with ARCA. CAP1 boosts translation compare to CAP0 in most cell types.

1. 2 – Pseudo-UTP and 5-Methyl-CTP to reduce immune response of cells against mRNA

We offer a variety of modified rNTPs suitable for in vitro transcription. This includes the Pseudo-UTP and 5-Methyl-CTP used to reduce cellular response against mRNA

It is now well established that a full substitution with these two modified rNTP reduces the innate immune response of cells. Such modRNA, as called by Pr Chien’s team in their article Nat. Biotechnol. in 2013 (10.1038/nbt.2682), can be used as treatment in vivo with no more need for HPLC purification

They showed that intramyocardial injection of VEGF-A coding modRNA after myocardial infarction (MI) induces vascular regenration (see picture below).

TriLink’s teams and partners have made further investigations and found that the 5-methoxy-UTP fully substitute mRNA can also efficiently reduce the immune response of the cells and the toxicity of the modified mRNA. The use of 5-methoxy-UTP (5moU) substitution is license free.

To track mRNA in vivo and optimise their delivery into cells, Cy5 labelling is possible though translation efficiency correlated inversely with Cyanine 5-UTP substitution.

Transcription with Cyanine 5-UTP:5-Methoxy-UTP at a ratio of 1:3. Substitution in this ratio results in mRNA that is easily visualized and can still be translated in cell culture.

Capped RNA Synthesis (E2040)

Introduction

The recommended ratio of cap analog to GTP is 4:1. Cap analogs are sold separately. Please refer to the ordering information section for more information.

a 4:1 mixture of a cap analog to GTP in transcription reactions will cap 80% of the resulting mRNAs

Protocol

1. Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.
2. Make 20 mM GTP solution by combining 2 μl of 100 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.
3. Prepare cap analog at the concentration of 40 mM.
4. Assemble the reaction at room temperature in the following order:

Nuclease-free water	X μl
10X Reaction Buffer	2 μl
ATP (100 mM)	2 μl	10 mM final
UTP (100 mM)	2 μl	10 mM final
CTP (100 mM)	2 μl	10 mM final
GTP (20 mM)	2 μl	2 mM final
Cap Analog (40 mM)	4 μl	8 mM final
Template DNA	X μl	1 μg
T7 RNA Polymerase Mix	2 μl
Total reaction volume	20 μl

1. Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours.
   
   The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used.
   
   Table 1. Effect of cap analog:GTP ratios on RNA yield

Cap Analog: GTP Ratio	Concentration of Cap Analog: GTP (mM)	RNA Yield (μg) in 2 hours	% Capped RNA
0:1	0:10	180	0
1:1	5:5	90–120	50
2:1	6.7:3.3	60–90	67
4:1	8:2	40–50	80
8:1	8.9:1.1	20–25	89

1. Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.
2. Proceed with purification of synthesized RNA or analysis of transcription productss by gel electrop to

Capped analog: Anti-Reverse Cap Analog (ARCA)

NEB: 3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog, S1411S, $151/µmol

https://www.trilinkbiotech.com/arca.html  ARCAN-7003, N-7003-1$142/ µmole 

https://www.trilinkbiotech.com/products-services/nucleoside-triphosphates-nucleotides/cap-analogs.html

mRNA Synthesis with Modified Nucleotides (E2060)

Modified mRNAs containing 5mCTP and Pseudo-UTP have been shown to suppress RNA-mediated innate immune activation in vivo. The HiScribe T7 ARCA mRNA Kit (with tailing) is capable of incorporation of 5mCTP and Pseudo-UTP, into mRNA. Up to 2.5 mM total modified nucleotides can be added into the transcription reaction without impacting the mRNA yield significantly. Other modified UTP or CTP may also be used but RNA yield will vary depending on the properties of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Please note modified nucleosides are not supplied with the kit.

The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP.

1. Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order:

Composition of NTP/cap analog mixture (

Component	Stock concentration (mM)	Final concentration (mM)	Volume (µl)
ATP (from MEGAscript T7 Kit)	75	7.5	4
GTP (from MEGAscript T7 Kit)	75	1.875	1
Me-CTP (from Trilink)	100	7.5	3
Pseudo-UTP (from Trilink)	100	7.5	3
3´-O-Me-m7G(5´)ppp(5´)G RNA cap structure analog	10	2.5	10
Total volume	23

IVT Reaction Mix

Component	Final concentration	Amount (µl)
Nuclease-free water	Variable
RNase Inhibitor	40 U	1
NTP/cap analog mixture (above)	23
PCR product	1 µg	Variable
10x reaction buffer	1x	2
10x T7 RNA polymerase enzyme mix	1x	2.5-4
Total volume	40

Nuclease-free water	to 20 µl
2X ARCA/NTP Mix	10 µl	1 mM GTP, 4 mM ARCA, 1.25 mM CTP, 1.25 mM UTP, >1.25 mM ATP final
5mCTP, 10 mM	2.5 µl	1.25 mM 5mCTP final
Pseudo-UTP, 10 mM	2.5 µl	1.25 mM Pseudo-UTP final
Template DNA	X µl	1 μg
T7 RNA Polymerase Mix	2 µl
Total reaction volume	20 µl

1. Follow standard mRNA synthesis protocol.

Capped RNA Synthesis (E2040)

Introduction

The recommended ratio of cap analog to GTP is 4:1. Cap analogs are sold separately. Please refer to the ordering information section for more information.

Protocol

Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

Make 20 mM GTP solution by combining 2 μl of 100 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.

Prepare cap analog at the concentration of 40 mM.

Assemble the reaction at room temperature in the following order:

Nuclease-free water	X μl
10X Reaction Buffer	2 μl
ATP (100 mM)	2 μl	10 mM final
UTP (100 mM)	2 μl	10 mM final
CTP (100 mM)	2 μl	10 mM final
GTP (20 mM)	2 μl	2 mM final
Cap Analog (40 mM)	4 μl	8 mM final
Template DNA	X μl	1 μg
T7 RNA Polymerase Mix	2 μl
Total reaction volume	20 μl

Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours.

The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used.

Table 1. Effect of cap analog:GTP ratios on RNA yield

Cap Analog: GTP Ratio	Concentration of Cap Analog: GTP (mM)	RNA Yield (μg) in 2 hours	% Capped RNA
0:1	0:10	180	0
1:1	5:5	90–120	50
2:1	6.7:3.3	60–90	67
4:1	8:2	40–50	80
8:1	8.9:1.1	20–25	89

Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.

Proceed with purification of synthesized RNA or analysis of transcription productss by gel electrop

1. Synthesize RNA using MEGAscript T7 kit (Ambion, Austin, TX)

1.1 Thaw the components from the T7 High Yield RNA Synthesis kit and keep on ice.

Component Volume (μl) Final

Nuclease free water* Xμl

10X Reaction Buffer 2μl

1X ATP (100 mM) 2μl 10mM

CTP (100 mM) 2μl 10mM

UTP (100 mM) 2μl  10mM

GTP (20 mM) 2μl  2mM

3 ́-0-Me-m7G(5')ppp(5')G cap analog (ARCA)(40mM) 4μl 8mM

Template DNA* Xμl 1μg

T7 RNA Polymerase Mix 2μl

Total 20 μl

Optionally:
5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies, San Diego, CA) are used to replace CTP and UTP.
Another protocol suggested to use 1.6 μg of purified tail PCR product as template in 40 μl reaction, and the final nucleotide reaction concentrations are 6 mM for the cap analog, 1.5 mM for guanosine triphosphate, and 7.5 mM for the other nucleotides.

1.2 Mix well by vortexing and incubate the reactions at 37 °C for 2 hours in a dry air incubator.

2. Removal of Template

DNA The transcription reactions are treated with DNase I (RNase-free) to remove the DNA template before proceeding with purification.

2.1. Add 70 μl nuclease free water to the transcription reactions followed by 10 μl of DNase I reaction buffer.

2.2. Add 2 μl DNase I to the reactions.

2.3. Incubate at 37 °C for 15 minutes in a dry air incubator.

3. Column Purification of Capped RNA

3.1. Purify the capped (or uncapped) RNA using MEGAclearKit or Qiagen RNeasy Mini Kit.

3.2. Quantify the RNA using a NanoDrop Spectrophotometer.

3.3. As per  manufacturer's instructions, assess RNA sample quality

(Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer was suggested but we do not have these).

4. RNA Capping using  Vaccinia Capping System

4.1. Use 10 μg of the purified capped mRNA. A standard capping reaction can cap up to 10 μg (100nt or larger) RNA. If a larger amount of RNA needs to be capped the reaction can be scaled up accordingly.

4.2. Increase the volume to 15 μl using nuclease free water.

4.3. Heat RNA solution at 65 °C for 10 minutes to remove secondary structures. Place on ice for 5 minutes.

4.4. To set up capping reaction, add 2 μl 10X Capping Buffer, 1 μl GTP (10 mM), 1 μl freshly diluted S-adenosyl methionine (2 mM) and 1 μl (10 units) Vaccinia capping enzyme to the 15 μl denatured RNA.

4.5. Mix well by gently vortexing and incubate  at 37 °C for 30 minutes in a dry air incubator.

4.6. Add 1.2 μl of 2 mM ZnCl₂ (20 X) to final conc of 0.1 mM,  or 2.3 μl 10X Antarctic Phosphatase Buffer, then add 1 μl of Antarctic Phosphatase and  incubate at 37 oC for 30 min.

5. Column Purification of Capped RNA

Purify capped RNA using MEGAclear Kit or Qiagen RNeasy Mini Kit

6. Quantitate RNA using Nanodrop and adjust to 100 ng/μL working concentration by addition of Tris-EDTA (TE, pH 7.0)

7. Assess RNA sample quality (such as Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer).

8. Store RNA at -80 oC.

2013-3-1

Lane 1: PCR   Lane 2:plasmid

Lane 1,2:PCR  Lane3:plasmid

DNA template Preparation

Plasmid with T7 promoter: linearize DNA using a restriction enzyme. Make sure the enzyme does not cut insert. Purify and measure DNA concentration.

Plasmid without T7 promoter: PCR insert using

F primer:

5' TAATACGACTCACTATAGGG--21 nt of gene specific sequence 3'

R primer: 5'- gene specific sequence--3'

Synthesis of Capped Cre Recombinase mRNA

This protocol describes the synthesis of Cre recombinase mRNA from a DNA template containing the Cre gene. Briefly, generic 5′ and 3′ UTRs are ligated to the Cre open reading frame (ORF). Ligation is facilitated by the use of splint oligonucleotides that bridge the junctions between the UTRs and the ORF. To this newly synthesized product, a tract of 120 Ts is appended by PCR. The final construct is amplified and used to template in vitro transcription of mRNA.

Materials

Cre ORF PCR

pBABE-puro-Cre (kindly provided by Dr. Zhe Li, Harvard Medical School)

KAPA HiFi Hotstart ReadyMix (Harvard Biopolymers – Catalog #KK2601)

5′ ORF Oligo - 5′TCCAATTTACTGACCGTACACC3′

3′ ORF Oligo - 5′CTAATCGCCATCTTCCAGCAGG3′

ORF Forward Primer Kinase Treatment

T4 Polynucleotide Kinase (PNK) with Buffer (New England Biolabs – Catalog #M0201)

Adenosine-5′-Triphosphate (ATP) (100 mM, USB (Affymetrix) – Catalog #77241)

5′ UTR and 3′ UTR Ligation

Cre Amplicon

5′ UTR Oligo

- 5′TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATG3′

3′ UTR Oligo, Phosphorylated

-5′TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATG3′

5′ Splint Oligos

- 5′GGTGTACGGTCAGTAAATTGGACATGGTGGCTCTTATATTTCTT3′

3′ Splint Oligos

- 5′CCCGCAGAAGGCAGCGATTAGCGGTAGAAGGTCGT3′

Ampligase Enzyme with Buffer (Epicentre Biotechnologies – Cat. No. A32750)

PCR to add poly-T tail (Tail PCR)

KAPA HiFi Hotstart Readymix (Harvard Biopolymers – Cat. No. KK2601)

Tail Forward Primer (Generic)

- 5′TTGGACCCTCGTACAGAAGCTAATACG3′

Tail Reverse Primer (Generic) PAGE Purified

- 5′T120CTTCCTACTCAGGCTTTATTCAAAGACCA3′

In Vitro Transcription

T7 MEGAscript Kit (Ambion – Catalog #AM1334M)

Adenosine-5′-Triphosphate (ATP) (100 mM, USB (Affymetrix) – Catalog #77241, 75 mM, included in T7 MegaScript Kit)

Guanosine-5′-Triphosphate (GRP) (100 mM, USB (Affymetrix) – Catalog #77243, 75 mM, included in T7 MegaScript Kit)

Pseudouridine-5′-Triphosphate (100 mM, TriLink Biotechnologies – Catalog #N-1019)

5-Methylcytidine-5′-Triphosphate (100 mM, TriLink Biotechnologies – Catalog #N-1014)

3′-O-Me-m7G(5′)ppp(5′)G Cap Analog (New England Biolabs – Catalog #S1411)

Tail PCR Products (100 ng/μL)

RNA Purification

MEGAclear Kit (Ambion – Catalog #AM1908M)

Phosphatase Treatment

Antarctic phosphatase (New England Biolabs – Catalog #M0289)

Cre Recombinase ORF PCR Amplification

1. Perform 5′ phosphorylation of the Cre ORF Forward primer.
Combine the following:
300 pmol primer (3 μL of 100 μL stock)
1 μL (10U) PNK enzyme
5 μL 10x buffer
50 nmol ATP (0.5 μL of 100 μL stock)
Incubate at 37 °C for 30 minutes
Heat-inactivate at 65 °C for 20 minutes
Add 250 μL TE to make 1 μM stock (100 μL to make 2 μM stock)

2. Amplify the Cre Recombinase ORF by PCR amplification. Perform a Gradient PCR around the Tm of the primers.
Perform 25 μL reactions by combining the following:
12.5 μL 2x KAPA HiFi Hotstart ReadyMix
3.75 μL Phosphorylated ORF Forward Primer (2 μM)
3.75 μL ORF Reverse Primer (2 μM)
Template DNA – 10–20 ng
H2O to bring final volume to 25 μl
Thermal cycler profile:
95 °C 5 min
98 °C 20 sec
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25 cycles
Gradient 15 sec
72 °C 60 sec
72 °C 5 min
4 °C Hold
Analyze on agarose gel (can use 1 μL of PCR reaction diluted 10x)

3. Perform a QIAQuick purification to purify the gel-excised DNA product (CRE recombinase). Use QIAQuick Spin Columns.
Combine 5 volumes of PB Buffer to 1 volume of reaction (5:1). Mix with pipettor and apply to column. Centrifuge at 6,000 rpm for 1 minute, and discard the flow-through.
Wash with 750 μL PE. Centrifuge at 6,000 rpm for 1 minute, and discard the flow-through.
Centrifuge again at 6,000 rpm for 1 minute and place filter on a clean Eppendorf tube.
Elute with water (nuclease-free) or EB buffer. Wait ≥ 1 minute before centrifuging at 6,000 rpm for 1 minute.

4. Quantify and quality control PCR product using Nanodrop machine.

UTR Ligation

5. Ligate the UTRs to the CRE recombinase ORF amplicon isolated above. Begin by combining the following for a 20 μL reaction:
4 μL 5′ UTR (1 μM)
4 μL 3′ UTR-Phosphorylated (1 μM)
2 μL 5′ Splint (1 μM)
2 μL 3′ Splint (1 μM)
2 μL 10x Buffer
1 μL Ampligase
1 μL CRE ORF amplicon
4 μl Water

6. Thermal profile for UTR Ligation:
95 °C 10 sec
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5 cycles
45 °C 60 sec
50 °C 60 sec
55 °C 60 sec
60 °C 60 sec
4 °C Hold

Perform QIAQuick Purification as described above in the Cre Recombinase ORF PCR amplification protocol.

NOTE: It may be desirable to TA clone the newly generated UTR-flanked Cre amplicon into a plasmid to avoid the need to re-amplify from the Cre DNA plasmid. This can be accomplished by using the pcDNA3.3-TOPO TA Cloning Kit.

NOTE: Check and ensure the ligation worked by diagnostic PCR by using flanking primers and subsequently performing gel electrophoresis to double-check if the product is of the expected size. This is a critical juncture for troubleshooting—if the ligation is unsuccessful (it often requires multiple attempts) try either a wider/smaller gradient of temperatures or try a sequential ligation. The difficulty lies in that the two splints usually have different annealing conditions so both will not stick at once.

Tail PCR

7. This section will append 120 Ts to the above synthesized Cre ORF with newly ligated 5′UTR and 3′UTR. Begin by diluting the PCR product (Cre ORF with 5′ UTR and 3′UTR) to 10 ng/μL.

8. Perform a 25 μL PCR reaction by combining the following:
12.5 μL 2x KAPA HiFi Hotstart ReadyMix
1 μL Tail Forward Primer (5 μM) – final concentration is 200 nM
1 μL Tail Reverse T120 Primer (5 μM) – final concentration is 200 nM
PCR product (10 ng/μL) – 10–20 ng
H2O to bring final volume to 25 μl

9. Thermal cycler profile for Tail PCR reaction:
95 °C 5 min
98 °C 20 sec
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25x
65 °C 15 sec
72 °C 45 sec
72 °C 10 min
4 °C Hold

10. Analyze the PCR product via gel electrophoresis (can use 1 μL of PCR reaction diluted 10x).

11. Perform QIAQuick Purification as described above in the Cre Recombinase ORF PCR amplification protocol.

12. Quantify and quality control the purification product by Nanodrop and dilute to 100 ng/μL

In Vitro Transcription of Modified Cre RNA

13. This section is for the in vitro transcription of modified Cre mRNA using the above stated protocol. Begin by preparing a nucleotide triphosphate (NTP) mix. This mix recipe is sufficient for 5.4 reactions and this proportion of Cap:GTP allows for ~80% of transcripts to be capped, though this can contribute to reduced yield. The cap powder should be dissolved in 35 μL H2O. The recipe is as follows:
Mix Concentration Desired Final Concentration
Cap 15 mM 6 mM
GTP 3.75 mM 1.5 mM
ATP 18.75 mM 7.5 mM
5methylCTP 18.75 mM 7.5 mM
PseudoUTP 18.75 mM 7.5 mM

14. For a 2x reaction (40 μL), combine reagents in the following order in a PCR strip at room temperature. The NTP Mix and template should be thawed on ice, then moved to room temperature (but only for setting up the IVT, immediately return to ice afterward). The 10x Buffer can be pre-warmed to 37 °C. It is important to ensure there is no precipitate in the 10x Buffer. The template concentration should be pre-adjusted at 100 ng/μl.
Custom NTP Mix 16 μL
10x Buffer (Kit) 4 μL
Template (Tail PCR) 16 μL (1.6 μg)
T7 Enzyme Mix (Kit) 4 μL
Total 40 μL

15. Incubate the reaction at 37 °C for up to 12 hours.

16. Perform DNase treatment with 2 μL TURBO DNase (Kit) for each reaction. Gently mix and incubate at 37 °C for 15 min.

17. Perform purification with MEGAclear Kit as described in the manufacturer’s instructions: Begin by pre-warming the 115 μL Elution Solution per reaction at 95 °C using PCR machine.

18. Bring the in vitro transcription (IVT) reaction volume to 100 μL by adding 58 μL of Elution Solution. Mix by pipetting and transfer the total volume to an RNase-free Eppendorf tube.

19. To the new RNase-free Eppendorf tube, add 350 μL Binding Solution Concentrate and mix by pipetting.

20. Add another 250 μL of 100% ethanol solution and mix by pipetting.

21. Transfer the sample to a filter cartridge (assembled on a collection tube).

22. Centrifuge for 1 min at 10,000 RPM. Take care to avoid the escape of glass fibers out of the column and into the collection tube. Discard the flow-through.

23. Wash each reaction with 500 μL Wash Solution and centrifuge for 1 min at 10,000 RPM. Discard the flow-through.

24. Repeat wash with 500 μL Wash Solution. Discard the flow-through.

25. Dry by centrifuging for 30 seconds at 10,000 RPM.

26. Place filter on a new collection tube and elute RNA by adding 50 μL of 95 °C Elution Buffer. Centrifuge for 1 min at 10,000 RPM.

27. Repeat the elution step with 50 μL of 95 °C Elution Buffer. Centrifuge for 1 minute at 10,000 RPM.

28. Quantify the RNA using a Nanodrop machine before proceeding to Antarctic phosphatase treatment and a second of purification by the MEGAclear Kit.

29. Perform Antarctic Phosphatase treatment by adding 10 μL 10x buffer to each reaction. Then add 3 μL Antarctic Phosphatase. Gently mix and incubate the reaction for 30 minutes at 37 °C. Optional: Inactivate reaction at for 5 minutes at 65 °C.

31. Perform RNA Purification with MEGAclear Kit as described above (Step 17 – 27).

32. Quantify the RNA using Nanodrop. Each 2x reaction will generally produce ~50 μg RNA (the expected yield of a 40 μL reaction is generally 40 – 60 μg).

33. Adjust the RNA concentration to 100 ng/μL using RNase-free TE buffer.

34. Aliquot RNA and store at −20 °C or −80 °C. −20 °C is preferable to reduce freeze/thaw cycles.

Note: To quality control RNA, load the RNA and run gel electrophoresis under native conditions. High quality RNA should result in the observation of a single band.

Cre RNA Transfection

1.  After allowing one or two days for recovery of pE4-hiPSCs from plating and single cell-dissociation, prepare to transfect with mRNA continuously for seven days.

2.  Dilute 100 ng/μl of Cre RNA 5x in Opti-MEM basal media. Dilute 5 μl of RNAiMAX per microgram of RNA 10x in Opti-MEM. Combine these two dilutions and incubate at room temperature for 15 minutes.

3.  Take the six-well plate(s) of pE4 hiPSCs out of the 37 °C incubator. Feed each well with 2 ml of fresh mTeSR1 media and 50 – 100 ng/ml of B18R interferon inhibitor.

4.  After changing the media, add RNA/RNAiMAX complex directly to the media.

5.  After 4 – 6 hours, refresh each well with 2 ml of mTeSR1 media and 50 – 100 ng/ml of B18R interferon inhibitor. All media changes should contain B18R when RNA transfection is occurring.

6.   Incubate the plates in the incubator overnight. and repeat this procedure (Step 11 – 14) every day for 7 days.

7.   After the seventh day of RNA transfection, allow putative Cre-excised hiPSC clones to expand into colonies. After observing colony formation, seed a 6-well gelatinized plate with CF1 MEF feeder cells.

Radioactive capped mRNA

5′ Cap analog (10 mM)
In addition to “conventional” cap analogs, which can be incorporated in two ways to produce mixtures of
differently capped mRNAs, Ambion sells an antireverse cap analog (ARCA), which ensures that the cap is in the
right orientation.
[α-32P]GTP (800 Ci/mmol; 10 μCi/μL) or [α-32P]UTP (800 Ci/mmol; 10 μCi/μL)
We recommend labeling capped transcripts with [α-32P]GTP because the concentration of GTP is already lowered for capping to be more efficient.
ATP, GTP, UTP, and CTP stocks (10 mM each, pH 7.0)
It is possible to initiate transcription with dinucleotides, i.e., UpG or CpG, or even mononucleosides (i.e., G).
Transcripts initiated in this way can be 5′ -end labeled without prior phosphatase treatment.
Dithiothreitol (DTT) (1 M)
DNase I (10 units/μL)
EDTA (0.5 M)
Ethanol (100%)
GlycoBlue (15 μg/μL)
Phenol:chloroform:isoamyl alcohol (PCA; 25:24:1)
RNasin (RNase inhibitor; 40 units/μL)
SDS extraction buffer <R>
Sodium acetate (3 M, pH 5.2)
T3 or T7 RNA polymerase (depends on template promoter; 50 units/μL)
Template DNA (linearized plasmid or gel-purified PCR product)
Transcription buffer for RNA polymerase (5×) <R>
Equipment
Dry ice
Gel-purification equipment
Incubator preset to 37 ̊C
Micro Bio-Spin Columns with Bio-Gel P-6 in Tris buffer (Bio-Rad)
Microcentrifuge
Microcentrifuge tubes (1.5 mL, polypropylene, capped)

 H2O to 50 μL

Template DNA (linearized plasmid or PCR product) 2 μg

0.04 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (10 mM) 2.5 μL

0.5 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/μL) 1 μL

0.8 unit/μL DTT (1 M) 1 μL

20 mM T3 or T7 RNA polymerase (50 units/μL) 1 μL 1 unit/μL 

To monitor the nonradioactive transcription reaction, remove 10 μL of the reaction and transfer to a separate tube. Add 1 μL of [α- 32P]GTP or UTP (800 Ci/mmol; 10 μCi/μL) and process in parallel with unlabeled GTP. For trace labeled, add 0.2–0.5 μCi of GTP or UTP (800 Ci/mmol). Remove and set aside
1 μL from any labeled reaction to use for input quantitation.
This reaction should yield  10–20 μg of RNA at 100 cpm/ng

For high-specific-activity RNA, combine the following in the order listed:

 H2O to 50 μL Template DNA (linearized plasmid or PCR product)

2 μg 0.04 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (0.5 mM) 2.5 μL

25 μM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/μL) 1 μL

0.8 unit/μL DTT (1 M) 1 μL

20 mM [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 10 μL

2 μCi/μL T3 or T7 RNA polymerase (depends on template promoter) (50 units/μL) 1 μL 1 unit/μL 

If labeling with [α- 32P]UTP, use 2.5 μL of 10 mM GTP (instead of 0.5 mM) and 2.5 μL of 0.5 mM UTP (instead of 10 mM) and add 10 μL of [α- 32P]UTP in the above reaction instead of [ 32P]GTP. Remove and set aside 1 μL to use for input quantitation. This reaction will yield 3 μg of RNA at 50,000–100,000 cpm/ng.

For moderate-specific-activity RNA, combine the following in the order listed:

 H2O to 50 μL

Template DNA (linearized plasmid or PCR product)

2–4 μg 0/04–0.08 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (0.5 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/μL) 1 μL 0.8 unit/μL DTT (1 M)

1 μL 20 mM [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 1–4 μL

1 unit/μL T3 or T7 RNA polymerase (depends on template promoter) (50 units/μL) 5 μL 1 μCi/μL

Adjust the amount of [α- P]GTP to get the desired specific activity. This reaction will yield RNA at 4000–8000 cpm/ng.

2. Incubate the reaction for 1–2 h at 37 ̊C. 3. Add 1 μL of 10 units/μL (for each 50 μL transcription reaction) DNase I and incubate for 15 min at 37 ̊C. 4. Add EDTA to 10 mM. 5. For labeled reactions, remove unincorporated nucleotides with a Micro Bio-Spin Column and then dilute to 200 μL with SDS extraction buffer. For unlabeled or trace-labeled reactions, dilute with an equal volume of SDS extraction buffer. Add 1/10 volume of 3 M sodium acetate (pH 5.2) to both labeled and unlabled reactions. 6. Extract with PCA (see Purification of RNA by SDS Solubilization and Phenol Extraction [Rio et al. 2010]). 7. Add 2 μL of 15 μg/μL GlycoBlue or glycogen to serve as carrier or to aid in resuspending the pellet. 8. Add 2.5 volumes of ethanol. Freeze on dry ice or in dry ice/ethanol bath ( 15 min). Thaw. Centrifuge at maximum speed in a microcentrifuge for 10 min. Carefully pipette off the supernatant and dispose off by an appropriate waste method.

9. Resuspend the pellets in 2 mM EDTA (2–5 μL for each 50 μL of transcription reaction for gel purification). If RNA is to be used directly, resuspend in H2O.

10. Gel-purify the RNA.

Check a small aliquot of longer transcripts (>600–700 nucleotides) on the gel for intactness. If the RNA is a single band with no trailing of radioactivity below, the transcripts can be used without gel purification.

Capping of In Vitro-Synthesized Transcripts For capping, mix the following reagents in the order listed:

Template DNA (linearized plasmid or PCR product) 2–4 μg

0.04–0.08 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (1 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/mL) 1 μL

0.8 unit/μL DTT (1 M)

1 μL 20 mM Cap analog (see above) (40 mM)

1.25 μL 1 mM T3 or T7 polymerase (50 units/μL)

1 μL 1 unit/μL [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 5 μL

1 μCi/μL H2O to 50 μL

Substitute any cap analog, dinucleotide (NpG), or GMP for 7mGpppG at a final concentration of 1 mM. 2. Perform Steps 2–10 above.

Random Incorporation of Modified Nucleotides during In Vitro Transcription The following is a “typical” in vitro transcription reaction designed to obtain a capped, moderate-specific-activity, biotin- or 4-thio-U-substituted RNA; the modified uridine is incorporated at approximately every fourth uridine residue. See Discussion.

1. For substitution, mix the following reagents in the order listed:

Template DNA (linearized plasmid or PCR product) 2–4 μg

0.04–0.08 μg/μL ATP (10 mM) 2.5 μL 0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 3.75 μL

0.375 mM Biotin-UTP or 4-thio-UTP (1 mM)

6.25 μL 0.125 mM GTP (1 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/mL) 1 μL

0.8 unit/μL DTT (1 M) 1 μL

20 mM Cap analog (see above) (40 mM) 1.25 μL

1 mM T3 or T7 polymerase (50 units/μL) 1 μL

1 unit/μL [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 5 μL

1 μCi/μL H2O to 50 μL

2. Perform Steps 2–10 above.

Problem (Step 9): The RNA does not completely resuspend. Solution: Resuspend the RNA in a larger volume or dilute it and reprecipitate with ethanol. Very efficient transcriptions may produce a PPi precipitate in large amounts and interfere with solubility. This problem can be overcome by addition of pyrophosphatase.

Problem (Step 10): The RNA yield is low. Solution: Rerun the reaction, making sure that the mixture remains in solution while assembling the reaction. The reactions are assembled at room temperature in Step 1 to avoid precipitation. If the reactions containing high concentrations of cap and nucleotides form a precipitate, heat them for 5 min at 37 ̊C and briefly vortex before adding the enzyme.

Problem (Step 10): The RNA is degraded. Solution: Make sure that the template was nuclease-free. If necessary, after plasmid linearization, dilute 100 μL of digestion with 200 μL of SDS extraction buffer. Add proteinase K (100 μg/mL), digest for 15 min at 37 ̊C, add 1/10 volume 3 M sodium acetate (pH 5.2) and proteinase K, phenol-extract, and ethanol-precipitate. Make sure that all solutions are RNase-free.

These methods are designed to prepare RNAs by in vitro transcription. Before setting up an in vitro transcription reaction, it is necessary to have a clear idea of how much RNA is needed and at what specific activity. In addition, is the RNA to be capped or substituted? Capping is usually performed in conjunction with radiolabeling and/or with other internal modifications. The presence of a cap at the 5′ end of in vitro-synthesized transcripts can be useful in many situations, e.g., by preventing 5′ to 3′ nuclease activity or by promoting translation. “Caps” come in several forms: 7mGpppG, ApppG, and GpppG. 7mGpppG serves as a recognition factor for the translation initiation factor eIF-4E and thus is important when making RNAs that will be used for translation. The other analogs are primarily used to prevent nucleolytic attack. Capping during transcription involves the initiation of RNA chains with the cap (obviously, cap cannot be incorporated internally). The extent of capping is determined by competition between cap and GTP;

therefore, cap must be present in excess of GTP. We recommend that the concentration of cap be 10-fold that of GTP; under such conditions, 90% of the RNAs will be capped. Because of constraints on the concentrations of cap that can be achieved (1 mM is practical), it is necessary to reduce the concentration of GTP to 100 μM to achieve efficient capping.

Many modified nucleotides can be incorporated into in vitro-synthesized RNA, including, but not limited to, biotinylated UTP, 4-thio-UTP, bromo-UTP, iodo-UTP, and many adenosine analogs (with α-thiophosphates). Biotinylated RNAs are used in affinity selection experiments; 4-thio-, 5-bromo-, and 5-iodo-U-substituted RNAs are useful for cross-linking studies; and α-thio-based analogs are primarily used for modification–interference experiments. When designing in vitro transcription reactions that incorporate modified nucleotides, it is important to consider the level of incorporation desired. For example, in modification–interference approaches, transcripts should contain one or even fewer modifications per transcript, whereas in biotin applications, heavier substitution (several per transcript) is desirable. The degree of substitution is determined by competition between the unmodified and modified nucleotide just as in capping. Because some modified nucleotides are incorporated less efficiently than their unmodified counterparts, the degree of substitution may not correspond exactly to the ratio of modified to unmodified nucleotides present in the transcription reaction. Thus, it is sometimes necessary to titrate the level of the modified nucleotide to achieve the desired level of substitution. Oversubstitution with biotin can sometimes cause RNA to partition with the organic phase because of the hydrophobic spacer. Transcripts heavily substituted with biotinylated nucleotides should not be extracted with phenol.

SDS Extraction Buffer

Reagent Quantity (for 500 mL)

Final concentration Tris–HCl (1 M, pH 7.5)

10 mL 20 mM EDTA (0.5 M, pH 8.0)

1 mL 1 mM SDS (10% w/v)

25 mL 0.5% (w/v) H2O 464 mL

Transcription Buffer for RNA Polymerase (5×)

Reagent Quantity (for 10 mL) Final concentration Tris–HCl (1 M, pH 8.0) 2 mL 200 mM MgCl2 (1 M) 400 μL 40 mM NaCl (5 M) 0.5 mL 250 mM Spermidine (100 mM) 1 mL 10 mM H2O 6.1 mL 

mRNA Precipitation

After adding 1U of RQ1 RNase-free DNase to the reaction and incubating at 37°C for 15 minutes, the RNA was precipitated by adding 30µl of nuclease-free water and 25µl of LiCl solution (7.5M lithium chloride, 50µM EDTA). The reaction was chilled for at least 30 minutes at –20°C and centrifuged (maximum speed in a microcentrifuge at 4°C for 15 minutes). The resulting RNA pellet was washed with 70% ethanol, dried and resuspended in 1mM sodium citrate (pH 6.4)

mRNA transfection

Cells were plated in 24 well tissue culture plates (Hela 50,000 cells per well; HepG2 100,000 cells per well) in MEM with 10% FBS prior to day of transfection. The following day cells were transfected with 125 ng of mRNA with 4 ul of Messenger Max (ThermoFisher Scientific, Waltham, MA) per well following the manufacture's protocol. Appropriate volume of mRNA was dissolved in OptiMEM to make up to 100 ul. All cells were harvested at 24 or 72 hrs post transfection and processed for protein analysis.