Invitrogen Corporation • 1600 Faraday Ave. • Carlsbad, CA 92008 USA • Tel: 760 603 7200 • FAX: 760 602 6500 • Toll Free Tel: 800 955 6288 • E-mail: tech_service@invitrogen.com • www.invitrogen.com

A Novel method to profile miRNA expression in different organisms:A Novel method to profile miRNA expression in different organisms:Sensitive and specific miRNA qRT-PCR with the NCode™ SYBR® Green miRNA Sensitive and specific miRNA qRT-PCR with the NCode™ SYBR® Green miRNA

qRT-PCR KitqRT-PCR Kit Sam An, Mark Landers, Mason Brooks, Pete Joszi, Ranjan J. Perera, and Christopher Adams

Invitrogen Corporation, Carlsbad California

MicroRNAs (miRNAs) are short 22-24 nucleotide RNAs that play a vital roles in regulating gene expression. These single stranded RNA molecules are incorporated into the RISC (RNA-inducing silencing complex) and regulate gene expression through various methods including translational inhibition, transcriptional cleavage and transcriptional gene silencing. Recent interest in these non-coding RNAs has been explosive because of its implication in cellular differentiation and disease genesis. Therefore, there is an urgent need to identify and validate miRNA expression in cell lines, tissues, and organs. qRT-PCR has become the standard for microarray data validation, as well as an invaluable tool for quantifying individual or subsets of miRNAs with the greatest sensitivity and accuracy. Most commercially available miRNA qRT-PCR systems employ proprietary, pre-designed miRNA-specific primers for cDNA synthesis by reverse transcription. Unfortunately, this approach requires that the sequence of the miRNA is publicly available and a commercial qRT-PCR assay has been developed for that specific sequence, limiting the availability of qRT-PCR assays for many model organisms as well as recently discovered miRNAs or proprietary miRNAs. Here, we describe a novel, universal, flexible and user-friendly qRT-PCR method, that was developed to measure and characterize miRNA expression in almost all organisms, starting from either total RNA and/or enriched miRNA. The NCode™ miRNA SYBR® Green qRT-PCR protocol is based on carefully optimized polyadenylation reaction with the reverse transcriptase, SuperScript™ III RT, in a “universal” 1st strand cDNA synthesis reaction.. The miRNA specific amplification occurs during the PCR reaction where the sequence of the miRNA of interest is used as the target-specific PCR primer. Platinum® SYBR® qPCR Supermix combines Platinum® Taq DNA polymerase with SYBR® Green fluorescent dye, delivers excellent sensitivity in the quantification of target sequences. This protocol offers 7 logs of dynamic range for maximum sensitivity (detection down to 100 copies) and single nucleotide discrimination for distinguishing between closely related miRNA families.

Abstract

Figure 1– NCodeTM miRNA qRT-PCR Overview

Figure 2– Assay Performance (Dynamic Range/Sensitivity)

Summary

Figure 3– Closely Related miRNA Family Discrimination

Figure 4– Validation of Microarray Data Through qRT-PCR

miRNA target

polyA tailing

Hybridization of tagged primer

Standard qPCR w/SYBR GreenHighly Flexible System

• Only 1 primer needed

• No special probes

• Quick assay design

• Permits analysis of other small RNAs

Accuracy over a broad dynamic range of samples

0.01

0.1

1

10

1 6 11 16 21 26 31 36 41

Cycle Number

Rela

tive E

xp

ressio

n (

? R

n)

100 million copies

100 copies

Slope = -3.51Y-int = 38.12R2 = 0.995

miRNA SequenceLet – 7a UGAGGUAGUAGGUUGUAUAGUULet – 7b UGAGGUAGUAGGUUGUGUGGUULet – 7c UGAGGUAGUAGGUUGUAUGGUULet – 7d AGAGGUAGUAGGUUGCAUAGU-Let – 7e UGAGGUAGGAGGUUGUAUAGU-

Let 7 Family Discrimination

0.01

0.1

1

10

Cycle Number (Ct)

Rel

ativ

e E

xpre

ssio

n

Let 7a Template(perfect match)

Let 7b - 7e(closely related mismatches)

The NCode™ SYBR® Green miRNA qRT-PCR Kit was used to profile synthetic let-7a-e miRNA templates from 100,000 copies using the let-7 A sequence as the primer. Using theannealing temperature of 65°C, the assay clearly discriminates the closely related family members. The closest mismatch (Let 7c) is approximately 10.5 CTs behind, which correlates to over 1000 fold difference in expression level.

Expression Level of miRNA in Specific Tissues (Microarray Data)

0

10000

20000

30000

40000

50000

60000

70000

mir-124a mir-128a mir-122a mir-194

miRNAs

Ex

pre

ss

ion

Le

ve

l

brain

liver

Primers Relative Expression in Brain Relative Expression in Liver

Mir-124A HIGHCt = 23.55

LOWCt = 31.79

Mir-128A HIGHCt = 25.15

LOWCt = 33.89

Mir-122A LOWCt = 35.56

HIGHCt = 21.55

Mir-194 LOWCt = 31.89

HIGHCt = 25.99

Published microarray data was verified through miRNA qRT-PCR. 4 pairs of miRNAprimers, 2 of which illustrated high expression level in either brain or liver tissuewere used to perform the qPCR. The qPCR results correlated identically with themicroarray data, as exhibited by CT values.

Amplification plot of a synthetic miRNA using the NCode™ SYBR® Green qRT-PCR Kit. The assay exhibits a broad dynamic range up to seven logs (100M to 100 copies) andshows an excellent linear correlation between copy number and CT value.

The NCodeTM miRNA qRT-PCR Assay offers unparalleled flexibility along

with a simple protocol to detect/quantitate miRNAs. No proprietary primers are

needed and can be used with any species on various small RNAs (incl. siRNAs,

snoRNAs, etc.)

The NCode™ miRNA qRT-PCR Assay provides 7 logs of dynamic range and

single nucleotide discrimination for maximum performance.

Primer Template CT

Let 7a 26.82Let 7b 38.21Let 7c 37.22Let 7d 38.6Let 7e 41.55NTC N/D

Let 7a