Editing MRNA by In vitro Transcription (section)

= mRNA Synthesis with Modified Nucleotides =

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

#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:<br/><br/>
{| border="0" width="100%" cellspacing="0" cellpadding="0"
|-
| Nuclease-free water
| to 20 µl
|-
| 2X ARCA/NTP Mix
| 10 µl
| 1 mM GTP, 4 mM ARCA,<br/>1.25 mM CTP, 1.25 mM<br/>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
|}

#Follow standard mRNA synthesis protocol.



== mRNA Purification ==

To harvest the RNA using Qiagen MinElute RNeasy Protocol For RNA cleanup:

#Adjust the sample to a volume of 100 uL with RNase-free water, add 350 uL of RLT and mix thoroughly (note: all older kits require the addition of β-ME to buffer RLT)
#Add 250 uL of ethanol (96-100&nbsp;%) to the diluted RNA, and mix thoroughly by pipetting. Do not centrifuge.
#Apply sample to an RNeasy mini column placed in a 2 mL collection tube. Close the tube gently, and centrifuge for 30 s at 13,200rpm. Discard flow-through and collection tube.
#Transfer the RNeasy column into a new 2 mL collection tube. Pipet 500 uL Buffer RPE onto the RNeasy column. Close tube gently, and centrifuge 1 minute for 13,200 rpm to wash the column and discard the flow-through.
#Add 500 uL of 80% Ethanol to RNeasy column. Close tube gently and centrifuge for 1 minute at 13,200 rpm. Discard flow-through and collection tube.
#Transfer the RNeasy column into a new 2 mL collection tube. Close tube gently and centrifuge for 5 minute at 13,200 rpm to dry the RNeasy silica-gel membrane.
#To elute transfer the RNeasy column to a new 1.5 ml collection tube. Pipet 50uL of RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube and centrifuge for 1 minute at 13,200 rpm.
#Immediately put collection tubes on ice, take 2 uL + 1.5ul of RNA out into two new 1.5 mL tubes.
#Quickly store the remaining RNA in -80℃.
#Run the taken-out 2 uL RNA on a gel and Quantify the 1.5 uL RNA using Nanodrop©!.
#Stored RNA can be diluted as necessary.
[[Category:Protocols]]