miScript PCR System

Overview

This protocol is used to generate and quantify cDNA from miRNA and mRNA in frozen tissue samples. Samples are prepared by immediate submersion in RNALater and incubated at room temp overnight, drained, snap frozen, and stored at -80ºC. RNA is extracted from these tissues using the Qiagen miRNeasy kit. This kit extracts total RNA, despite it's name. This total RNA is used as input for the miScript II RT Kit, which will convert the RNA to cDNA. The kit has two buffers, we use the HiFlex Buffer, which is designed to capture both miRNA and mRNA. The cDNA generated from the miScript II RT Kit is then split into two aliquots. One aliquot is used for detecting and quantifying mature miRNA, while the second aliquot is used for detecting and quantifying mRNA. To quantify mature miRNA, we use the miScript Primer Assay Kit, followed by the miScript SYBR Green PCR kit. For detecting an quantifying mRNA, we use the QuantiTect Primer Assay, followed by the QuantiTect SYBR Green PCR Master Mix (provided in the miScript SYBR Green PCR kit).

Protocol

Design of forward primers for miRNA quantification

Generation of cDNA using miScript II RT Kit

• Set up all reactions on ice, keep everything cold to reduce RNA degradation.
• Do not vortex template RNA or any of the components of the miScript II RT kit
• Thaw RNA on ice, while other reagents can be thawed at room temp. Remove miScript Reverse Transcriptase Mix from -20 just before using and return right after using, otherwise the enzyme will die.
• Aliquot RNA template into PCR tubes or plates. Each tube should contain 12 µl volume of RNA template. Remember to prepare at least one tube for the no RT control. Each tube should have between 10 pg and 1 µg of template RNA in 12 µl of volume. Use RNase-free water for diluting. Ideally, all tubes will have roughly the same total amount of RNA.
• Prepare two master mixes on ice (one with Reverse Transcriptase and one without Reverse Transcriptase). Make sure the master mixes have enough volume for all the samples (add 10% extra). Each tube will require 8 µl of the master mix.
• Prepare the reverse transcription master mix on ice, multiplying the amounts below by the number of samples and then adding 10%:
  • 4 µl 5x miScript HiFlex buffer
  • 2 µl 10x miScript Nucleics Mix
  • 2 µl miScript Reverse Transcriptase Mix
• Prepare the NO reverse transcription master mix on ice, multiplying the amounts below by the number of samples that you are using for no RT controls and then adding 10%:
  • 4 µl 5x miScript HiFlex buffer
  • 2 µl 10x miScript Nucleics Mix
  • 2 µl RNase free water
• Add 8 µl of the master mix to each of the samples, gently mix and centrifuge. At this point, samples should be stored on ice
• Incubate tubes/plates at 60 min for 37ºC in a PCR machine, followed by 5 min at 95ºC to inactivate the Reverse Transcriptase enzyme. At this point the cDNA can be stored at -20ºc either in undiluted or diluted aliquots.
• Quantify the cDNA using a nanodrop machine. For parallel detection of mature miRNA and mRNA, you will need to add 10-20 ng of cDNA in a volume of 2.5 µl or less for each PCR reaction.

Quantitation of miRNA

• Prepare