ReverseTranscription-Protocol-miRNA-SCALONMC
Marcela Scalon, Christine Souza Martins, Gabriel Ginani Ferreira, Franciele Schlemmer, Ricardo Titze de Almeida, Giane Regina Paludo
Abstract
This protocol is intended as a guideline to perform the Reverse Transcription procedure to detect and quantify miRNAs. This method use TaqMan® MicroRNA Assays and Applied Biosystems real-time PCR instruments. The assays can detect and quantify small RNA in 1 to 10 ng of total RNA with a dynamic range of greater than six logs. This protocol works on detecting microRNA from samples that were purified from plasma and serum. All samples were stored at -80ºC until the realization of the RT procedure. The aim of this protocol is to use a miRNA-specifc, stem-loop RT primer, in order to produce cDNA from miRNA samples. The amplified products of RT procedure will then be used to perform qPCR or rtPCR.
Before start
Just before RT, and right after thawing of the samples, the step of quantification must be performed. Within the concentration values of each sample in hand, it is necessary to dilute all samples to the lowest value in order to level all concentrations that will run in the same set.
Allow the kit components to thaw on ice.
Steps
Prepare de RT mix
Allow the reagents and samples to thaw on ice.
Before use, mix gently and centrifuge briefly.
In a polypropylene 0,6 tube, prepare the RT mix on ice by scaling the volumes listed below to the desired number of RT reactions + 1 (excess volume to compensate for losses that occur during pipetting):
| A | B |
|---|---|
| Component | Mix volume per 15-μL reaction* |
| Nuclease-free water | 4.16 μL |
| 100mM dNTPs (with dTTP) | 0.15 μL |
| MultiScribe™ Reverse Transcriptase, 50 U/μL | 1.00 μL |
| 10✕ Reverse Transcription Buffer | 1.50 μL |
| RNase Inhibitor, 20 U/μL | 0.19 μL |
| Total volume | 7.00 μL |
- Each 15-μL RT reaction consists of 7 μL master mix, 3 μL of 5X RT primer, and 5 μL RNA sample.
Mix gently. Centrifuge to bring the solution to the bottom of the tube.
Place the RT mix on ice until you prepare the RNA reaction.
Prepare the RT reaction
Thaw the 5✕ RT primer from the TaqMan® MicroRNA Assays (target and control) on ice.
Thaw the diluted samples (2 ng/μL) on ice.
Before use, vortex the RT primer tubes to mix, then centrifuge briefly.
Prepare a 0.2 ml tube for each sample.
Label the lid of the tube.
For each 15-μL RT reaction, combine RT mix (from step 2) with miRNA sample and 5X RT primer in the ratio of:
-
7 μL of RT master mix
-
5 μL of miRNA sample (2 ng/μL per reaction)
-
3 μL of 5X RT primer from each assay into the corresponding RT reaction tube.
Note: Before opening the RT primer tubes, thaw the tubes on ice and mix by vortexing, then centrifuge them.
Seal the tubes and mix gently.
Centrifuge briefly to bring the solution to the bottom of the tube.
Incubate the tube on ice for 5 minutes and keep it on ice until you are ready to load the thermal cycler.
Perform reverse transcription (RT)
Note: If applicable, perform the reverse transcription in Standard mode.
Use the following parameter values to program the thermal cycler:
| A | B | C |
|---|---|---|
| Step | Time | Temperature |
| Hold | 30 minutes | 16 °C |
| Hold | 30 minutes | 42 °C |
| Hold | 5 minutes | 85 °C |
| Hold | ∞ | 4 °C |
Set the reaction volume to 15.0 μL.
Load the reaction tube or plate into the thermal cycler.
Start the RT run.
Note: If you do not immediately continue to PCR amplification after the RT run, store the RT reaction at − 15 to − 25 °C.
