Editing Capped mRNA (section)

== 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.<br/><br/>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.<br/><br/>Table 1. Effect of cap analog:GTP ratios on RNA yield

{|
|-
| width="25%" | '''Cap Analog: GTP Ratio'''
| width="25%" | '''Concentration of Cap'''<br/>
Analog: GTP (mM)

| width="25%" | '''RNA Yield (μg) in'''<br/>
2 hours

| width="25%" | '''% 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'''&nbsp; 10mM

GTP (20 mM) '''2μl'''&nbsp; 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:'''<br/>5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies, San Diego, CA) are used to replace CTP and UTP.<br/>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.

<br/>'''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.

<br/>'''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&nbsp; 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&nbsp; 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&nbsp; at 37 °C for 30 minutes in a dry air incubator.



4.6. Add 1.2 μl of 2 mM ZnCl<sub>2</sub> (20 X)&nbsp;to final conc of 0.1 mM,&nbsp; or 2.3 μl 10X&nbsp;Antarctic Phosphatase Buffer, then add 1 μl of Antarctic Phosphatase and&nbsp; incubate at 37 oC for 30 min.

<br/>'''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&nbsp;&nbsp; Lane 2:plasmid

[[File:RNA 3-1-2013.JPG|RTENOTITLE]]

Lane 1,2:PCR&nbsp; Lane3:plasmid [[File:Capped mRNA 3-1-2013.JPG|RTENOTITLE]]