QIAGEN RNA User Manual

Sample to Insight__

April 2020

RNA Spike-in Kit, for RT,
Handbook

For controlling the quality of RNA isolation,
cDNA synthesis and PCR amplification for
miRCURY® LNA® miRNA PCR and miRCURY
LNA miRNA Probe PCR experiments

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RNA Spike-in Kit, for RT, Handbook 04/2020

Contents

Kit Contents................................................................................................................ 3

Shipping and Storage ................................................................................................. 4

Intended Use .............................................................................................................. 4

Safety Information ....................................................................................................... 4

Quality Control ........................................................................................................... 4

Introduction ................................................................................................................ 5

Important Notes .......................................................................................................... 7

Equipment and Reagents to Be Supplied by User ............................................................ 8

Protocol: Using the UniSp2, UniSp4, UniSp5 RNA Spike-in Mix in RNA Isolation ............... 9

Protocol: Using the UniSp6 and cel-miR-39-3p RNA Spike-ins in cDNA Synthesis ............. 12

Analysis and Interpretation of Data ............................................................................. 13

Ordering Information ................................................................................................ 20

Document Revision History ......................................................................................... 23

RNA Spike-in Kit, for RT, Handbook 04/2020

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Kit Contents

RNA Spike-in Kit, for RT
Catalog no.
Number of reactions

UniSp2, UniSp4, UniSp5 RNA Spike-in template mix:

Synthetic UniSp2 RNA (22 nt), 160 fmol

Synthetic UniSp4 RNA (22 nt), 1.6 fmol

Synthetic UniSp5 RNA (22 nt) , 0.016 fmol

MS2 total RNA, 50 ng

cel-miR-39-3p RNA Spike-in template:

Synthetic cel-miR-39-3p RNA, 0.16 fmol

MS2 total RNA, 50 ng

Quick-Start Protocol 1

339390
50

Conc. (resuspended):

2 fmol/µl

0.02 fmol/µl

0.00002 fmol/µl

0.625 ng/µl

0.002 fmol/µl

0.625 ng/µl

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RNA Spike-in Kit, for RT, Handbook 04/2020

Shipping and Storage

The RNA Spike-In Kit, for RT, is shipped at room temperature with the RNA content dried down.
Upon arrival the kit should be stored at –15 to –30ºC. Under these conditions, all components
are stable until the expiration date on the vial. It is recommended to store the RNA Spike-ins
in aliquots at –15 to –30ºC after resuspension to avoid repeated freeze-thaw cycles.

Intended Use

The RNA Spike-In Kit, for RT, is intended for molecular biology applications. These products
are not intended for the diagnosis, prevention or treatment of a disease.

All due care and attention should be exercised in the handling of the product. We recommend

®

all users of QIAGEN
recombinant DNA experiments, or to other applicable guidelines.

products to adhere to the NIH guidelines that have been developed for

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit
and kit component.

Quality Control

In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of RNA
Spike-In Kit, for RT, is tested against predetermined specifications to ensure consistent
product quality.

RNA Spike-in Kit, for RT, Handbook 04/2020

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Introduction

The primary purpose of the RNA Spike-in Kit, for RT, and the matching miRCURY LNA miRNA
PCR Assays or miRCURY LNA miRNA Probe PCR Assays for detecting the RNA spike-ins (see
Table 1 and below) is to provide a control for the quality of the RNA isolation, cDNA synthesis
and PCR amplification for miRCURY LNA miRNA PCR or miRCURY LNA miRNA Probe PCR
experiments. This will help to identify experimental and technical outliers.

Table 1. LNA PCR assays for the RNA Spike-in Kit templates

Corresponding miRCURY LNA miRNA PCR Assay

Spike-in template Catalog number Product number

Synthetic UniSp2 RNA 339306 YP00203950

Synthetic UniSp4 RNA 339306 YP00203953

Synthetic UniSp5 RNA 339306 YP00203955

cel-miR-39-3p RNA 339306 YP00203952

Table 2. LNA PCR Probe assays for the RNA Spike-in Kit templates

Corresponding miRCURY LNA miRNA Probe PCR Assay

Spike-in template Catalog number Product number

Synthetic UniSp2 RNA 339350 ZP00004671

Synthetic UniSp4 RNA 339350 ZP00004673

Synthetic UniSp5 RNA 339350 ZP00004687

cel-miR-39-3p RNA 339350 ZP00004672

Reproducible RNA isolation may be difficult from some sample types, and some RNA samples
may contain inhibitors of cDNA synthesis or PCR, even though they were isolated using the
best standard procedures. This can lead to different efficiencies of the reverse transcription or
PCR amplification between compared samples. One way to check for differences in
efficiencies in isolation, cDNA synthesis and PCR amplification is by adding known amounts

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RNA Spike-in Kit, for RT, Handbook 04/2020

of RNA spike-ins to the sample prior to RNA isolation and cDNA synthesis, respectively. Use
of RNA spike-ins may also reveal potential presence of nucleases.

After conducting the PCR, but before progressing into data analysis, wells detecting the RNA
spike-ins are compared, and outlier samples may be identified and considered for exclusion
from further analysis. The RNA Spike-in Kit, for RT, provides several RNA spike-ins. Three of
the RNA spike-in templates (UniSp2, UniSp4 and UniSp5) are provided premixed in one
vial, each at a different concentration in 100-fold increments. This mix is meant as an RNA
isolation control.

A second vial contains a synthetic version of a

C. elegans

miRNA, cel-miR-39-3p. The cel-miR­39-3p RNA spike-in template is meant to be used in combination with the UniSp6 RNA spike­in template provided with the miRCURY LNA RT Kit (cat. no. 339340). This mix is meant as a
cDNA synthesis control.

The UniSp6 RNA spike-in control PCR assay can be found in all of the predefined miRCURY
LNA miRNA PCR Panels and miRCURY LNA miRNA Probe PCR Panels. When configuring
custom PCR panels, it is possible to select from all five control PCR assays in the plate layout.

All of our predefined and custom miRCURY LNA miRNA PCR Panels and miRCURY LNA
miRNA Probe PCR Panels include a special interplate calibrator. In the miRCURY LNA miRNA
PCR Panels, it is a triplicate set of UniSp3 assays with template; in the miRCURY LNA miRNA
PCR Probe Panels, it is a triplicate set of UniSp9 assays with template for each plate. These
interplate calibrators can also be used for PCR amplification controls.

RNA Spike-in Kit, for RT, Handbook 04/2020

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Important Notes

The synthetic RNA spike-ins in the PCR Spike-in Kit, for RT, are controls for RNA isolation,
cDNA synthesis and PCR efficiency. They should be used for checking that these technical
steps have worked properly, but they should never be used for normalization. Synthetic RNA
spike-ins do not reveal the RNA content and quality in the biological sample. Normalization
should always be performed using stably expressed endogenous reference genes or, when
applicable, the global mean of all expressed miRNAs.

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RNA Spike-in Kit, for RT, Handbook 04/2020

Equipment and Reagents to Be Supplied by User

When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, consult the appropriate safety data sheets (SDSs)
available from the product supplier.

For use with the miRCURY LNA miRNA PCR System

 miRCURY LNA SYBR

 miRCURY LNA RT Kit (cat. no. 339340)

 Individual miRCURY LNA miRNA PCR Assays or Panels

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Green PCR Kit (cat. nos. 339345, 339346, 339347)

For use with the miRCURY LNA miRNA Probe PCR System

 miRCURY Probe PCR Kit (cat. nos. 339371, 339372, 339373)

 miRCURY LNA RT Kit (cat. no. 339340)

 Individual miRCURY LNA miRNA Probe PCR Assays or Panels

For use with both systems

 Individual PCR assays for the various RNA Spike-in Kit templates (see Table 1 or Table 2)

 Nuclease-free PCR tubes or plates for use with individual assays

 Nuclease-free, aerosol-barrier pipette tips

 Nuclease-free, low nucleic acid binding (siliconized) microcentrifuge tubes

 Sealing foil for PCR plates

 Microcentrifuge and plate centrifuge

 Heating block, thermal cycler or other incubators

 Real-time PCR instrument