Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Summary

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Abstract

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels. One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay. Automated qPCR setup has improved this field by allowing for greater reproducibility. Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment. This method along with internal plate controls also reduces experimental variables common to other techniques. We recently developed a qPCR assay for profiling of pre-microRNAs (pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level. These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful. There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique. Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing. Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Protocol

The qPCR pre-miRNA profiling arrays can be set up as fully automated with a Tecan Freedom Evo robot (A) or by hand with the Matrix electronic multichannel pipette (B).

1)Prepare the master mix, primer plates and samples.

1. Primer plates containing 186 primer pairs in 96-well format (total of 2 plates) at 0.5pM should be stored at -80°C. Thaw plates out at room temperature, vortex and centrifuge briefly before use.
2. To prepare the master mix, thaw out SYBR Green 2x PCR Mix at room temperature. Each reaction uses 8ul master mix with 2ul primer. The composition of the master mix per reaction is 4ul SYBR mix, 3ul PC....

Discussion

QPCR is a highly sensitive assay that can be used to compare gene expression levels between samples in a study, as well as to determine positivity in the case of viruses. The benefit of using qPCR arrays is the ability to run many primers (in our case, 186 primer pairs) for each sample in a short period of time. Using a pipetting robot such as the Tecan Freedom Evo, the amount of time required to set an experiment up can be further reduced, and the accuracy and consistency of the robot can reduce and eliminate pipettin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Freedom Evo 150	Tecan Group Ltd.	30017587
Matrix Electronic Multichannel Pipette	Thermo Fisher Scientific, Inc.	2001-MTX	(or any comparable Thermo Scientific multichannel that can pipette the volumes described above)
Matrix Integrity Filter Tips, Sterile	Thermo Fisher Scientific, Inc.	7435	(or comparable tip for multichannel used)
Lightcycler 480 SYBR Green 1 Master	Roche Group	04 707 516 001
Lightcycler 480 Instrument II	Roche Group	05015243001	384-well version
Lightcycler 480 Multiwell Plate 384, white	Roche Group	04729749001
LightCycler 480 Sealing Foil	Roche Group	04729757001
LightCycler 480 Multiple Plate Analysis Software	Roche Group	05075122001
Eliminase Decontaminant	Decon Laboratories	04-355-31