To convert RNA into cDNA. You will use a protein called Reverse Transcriptase, which is a polymerase that synthesizes DNA from RNA. The reaction requires primers, which can be either oligodT (annealing to polyA tails of mRNA) or random hexamers; the kit used here contains both kinds); nucleotides for DNA synthesis (dNTPs); MgC2 and buffers required by the enzyme, which are all present in the 5x Reaction Mix.
* iScript cDNA Synthesis Kit (BIORAD Cat. No. 170-8891)
- 5x iScript Reaction Mix
- Nuclease-free water
- iScript Reverse Transcriptase (RT)
* Waterbaths: - 25°C
*Alternatively, you can use the Thermocycler, using the following program:
* 1.5ml Nuclease-free tubes
* RNaseZap (Ambion Cat. No. 9780)
- Clean bench top, pipettes, racks, with RNaseZap: spray it on everything and leave on for a few minutes, then remove carefully with Kim wipe. Then spray all with 70% Ethanol. Clean your gloves the same way.
- This protocol is optimized for up to 1µg RNA. Used the volume of RNA that contains this amount, or less. If you need to RT more RNA, multiply the reaction volumes by the appropriate factor, up to 5µg RNA.
1µg RNA/ [RNA] µg/µl = V(µl) RNA containing 1µg
- If you have DNase-treated the RNA with Invitrogen, then the volume containing 1µg must be 11µl. However, sometimes there is a little evaporation, so you need to measure and record the volume in each sample before starting. Spin down tube first to get maximum recovery of sample.
- You need to have at least one – RT Control. This control contains everything in the reaction (RNA, buffers, water) except for RT protein. Replace volume of RT with same volume of water.
- Sample calculations for RT reaction are shown below.
1. Add RNA
a. Add RNA to an amount of 1mg RNA (for a reaction volume of 20ml)
Note: The amount of RNA that can be used for this rxn volume is 100fg to 1mg RNA; if more RNA is used, (e.g. 2mg), add reagents to a factor, (e.g. to a 40ml reaction volume).
b. Place tube immediately on ice. Remove tube only to add components, one at a time, and place back on ice.
2. Add Nuclease-free water to 15ml
3. Add 5x iScript Reaction Mix, 4ml for a 20ml rxn volume.
4. Add iScript RT, 1ml.
5. Incubate complete reaction mix at following temperatures:
a. 5 minutes at 25°C
b. 30 minutes at 42°C
c. 5 minutes at 85°CStore samples at –20ºC