Aug 23, 2024
High-Capacity cDNA Reverse Transcription
- 1University of Minnesota
- Team Lee
Protocol Citation: Hector Martell Martinez 2024. High-Capacity cDNA Reverse Transcription. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8wq2lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2024
Last Modified: August 23, 2024
Protocol Integer ID: 106358
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000592
Abstract
This protocol details the high-capacity cDNA reverse transcription.
Materials
- Applied BiosystemsTM High-Capacity cDNA Reverse Transcription KitApplied Biosystems (ThermoFisher Scientific)Catalog #4368814
Master Mix:
| A | B | |
| Component | Volume | |
| 1) Nuclease-free Water | 3.2 µL | |
| 2) 10X RT Buffer | 2.0 µL | |
| 3) 10X Random Primers | 2.0 µL | |
| 4) RNase Inhibitor | 1.0 µL | |
| 5) 25X dNTP Mix | 0.8 µL | |
| 6) MultiScribe Reverse Transcriptase | 1.0 µL ***add last!! | |
| TOTAL per reaction | 10 µL |
Nanodrop
Nanodrop
Nanodrop each isolated sample of RNA.
A good concentration of RNA is between 200 undetermined - 2000 undetermined .
- If the concentration is above 2000 undetermined then dilute the sample with water for a final concentration below2000 undetermined .
A good 260/280 value is ~2.0.
A good 260/230 value is ~2.0-2.2.
cDNA Calculations
cDNA Calculations
cDNA for all brain regions is made with 2000 ng of RNA. cDNA for isolated cells is made with the highest amount of RNA that can be made from the least concentrated sample.
TV per reaction = 20 µL (10 µL of RNA/Water + 10 µL of Master Mix)
- Calculate the RNA amount needed to make 2000 ng of RN for each sample
Ex. For a RNA concentration of 1250.0 undetermined .
2000/1250 = 1.6 µL of RNA
Calculate the amount of water to be added to the RNA for a TV = 10 µL
Ex. For 1.6 µL of RNA
10 µL – 1.6 µL of RNA = 8.4 µL of Water
Making cDNA
Making cDNA
Thaw the isolated RNA and the following components of the High-Capacity cDNA reverse transcription kit On ice .
- 10X RT Buffer
- 25X dNTP Mix (100 millimolar (mM) )
- 10X Random Primers – Can thaw at Room temperature
- RNase Inhibitor
Note
- DO NOT thaw MultiScribe Reverse Transcriptase – it does not freeze at -20 °C and is prone to denaturing at higher temperatures. Keep at -20 °C until creating your master mix in below.
While the above components thaw On ice pipette the calculated amount of water (from of cDNA calculations) to PCR tubes. This step can be done at Room temperature .
Place the PCR tubes with water On ice and then add the calculated amount of RNA (from of cDNA calculations) to its respective PCR tube.
Create the following master mix On ice . Make enough master mix for each sample plus a little extra (if you have 10 samples, make enough master mix for 11).
- Add reagents to a 1.5 mL tube in the following order.
| A | B | |
| Component | Volume | |
| 1) Nuclease-free Water | 3.2 µL | |
| 2) 10X RT Buffer | 2.0 µL | |
| 3) 10X Random Primers | 2.0 µL | |
| 4) RNase Inhibitor | 1.0 µL | |
| 5) 25X dNTP Mix | 0.8 µL | |
| 6) MultiScribe Reverse Transcriptase | 1.0 µL ***add last!! | |
| TOTAL per reaction | 10 µL |
Mix gently.
Add 10 µL of master mix to each PCR tube On ice .
Mix PCR tubes gently then spin down briefly.
Keep On ice until performing the reverse transcription.
Perform Reverse Transcription
Perform Reverse Transcription
Place PCR tubes into the thermal cycler.
Set the Reaction Volume to 20 µL .
Set the following conditions:
Start the thermal cycler run.
When the samples reach take the PCR tubes out and store at 4 °C for short term use and at -20 °C for long term use.
Protocol references
Refer to the applied biosystems “High Capacity cDNA Reverse Transcription Kit User Guide” for reference.