DESCRIPTION Template RNA CONTROL REACTIONS for RT-PCR / RT-qPCR

The Thermo Scientific™ Maxima™ H Minus First Strand Total cellular RNA isolated by standard methods is The following negative control reactions should be used to
cDNA Synthesis Kit is a complete system for efficient suitable for use with the kit. Purified RNA must be free of verify the results of the first strand cDNA synthesis.
synthesis of first strand cDNA. The kit uses the Maxima H salts, metal ions, ethanol and phenol to avoid inhibiting the • Reverse transcriptase minus (RT-) negative
PRODUCT INFORMATION Minus Reverse Transcriptase (RT), an advanced enzyme cDNA synthesis reaction. control is important in RT-PCR and
derived by in vitro evolution of M-MuLV RT. The enzyme For RT-PCR applications, template RNA must be free of RT-qPCR reactions to assess for genomic DNA
Thermo Scientific DNA contamination. Prior to cDNA synthesis, RNA can be
features the highest thermostability among the derivatives contamination of the RNA sample. The control RT-
Maxima H Minus of M-MuLV RT and lacks the RNase H activity. The treated with DNase I, RNase-free (#EN0521) to remove reaction should contain all reagents for the reverse
Maxima H Minus First Strand cDNA Synthesis Kit is trace amounts of DNA. transcription reaction except the Maxima H Minus
First Strand cDNA capable of synthesizing cDNA up to 20 kb from a wide Always perform a RT-minus negative control reaction, Enzyme Mix.
which includes all components for RT-PCR except the
Synthesis Kit range of total RNA amounts (1 pg to 5 µg) at elevated
Maxima H Minus Enzyme Mix.
• No template control (NTC) is important to assess
temperatures (45-65 °C) superceeding other systems in for reagent contamination. The NTC reaction should
#K1651 20 rxns ability to prepare full length cDNA. Due to increased RNA sample quality contain all reagents necessary for the reverse
Lot __ Expiry Date __ synthesis rate reaction can be completed in 30 min. Assess RNA integrity prior to cDNA synthesis. Total transcription reaction except the RNA template.
Maxima H Minus Enzyme Mix contains Maxima H Minus eukaryotic RNA can be analyzed by agarose gel PROTOCOLS
Store at -20°C Reverse Transcriptase and Thermo Scientific™ electrophoresis followed by ethidium bromide staining. The Please read the IMPORTANT NOTES section before

d
RiboLock™ RNase Inhibitor. The recombinant RiboLock RNA is considered to be intact, if both 18S and 28S rRNA starting.
RNase Inhibitor effectively protects RNA from degradation appear as sharp bands after electrophoresis of total RNA.
by RNases A, B and C at temperatures up to 55 °C. RT-PCR
The 28S rRNA band should be approximately twice as I. First Strand cDNA Synthesis
The kit is supplied with both oligo(dT)18 and random intense as the 18S rRNA. Any smearing of rRNA bands is
www.thermoscientific.com/onebio hexamer primers. Random hexamer primers bind non- After thawing, mix and briefly centrifuge the components of
an indication of degraded mRNA. If this occurs, a new the kit. Store on ice.
specifically and are used to synthesize cDNA from all sample of total RNA should be prepared. Alternatively,
RNAs in total RNA population. The oligo(dT)18 primer 1. Add the following reagents into a sterile, RNase free
COMPONENTS OF THE KIT total RNA can be analyzed using a bioanalyzer (e.g., tube on ice in the indicated order:
Maxima H Minus First #K1651 #K1652 selectively anneals to the 3’-end of poly(A) RNA, Agilent 2100) which provides quantitative information
Strand cDNA Synthesis Kit 20 rxns 100 rxns synthesizing cDNA only from poly(A) tailed mRNA. Gene- about the general state of the RNA sample, the RNA Template total RNA 1 pg - 5 µg
Maxima H Minus Enzyme Mix 25 µL 120 µL specific primers may also be used with the kit to prime integrity number (2). A reference gene/target gene 3’:5’ RNA or poly(A) mRNA 0.1 pg – 500 ng
5X RT Buffer synthesis from a specified sequence. integrity assay (3) can also be used to determine the or specific RNA 0.01 pg - 500 ng
250 mM Tris-HCl (pH 8.3 at 25°C), 150 µL 500 µL The synthesized cDNA can be used directly in PCR with a integrity of the RNA sample.
375 mM KCl, 15 mM MgCl2, 50 mM DTT Primer oligo (dT)18 1 µL (100 pmol)
variety of thermostabile DNA polymerases, in qPCR with
10 mM dNTP Mix 50 µL 250 µL RNA quantity primer
Thermo Scientific Maxima qPCR Master Mixes or in
Oligo(dT)18 Primer, 100 µM 25 µL 120 µL Use 1 pg - 5 µg of total RNA, 0.1 pg - 500 ng of or random 1 µL (100 pmol)
second strand cDNA synthesis. •
Random Hexamer Primer, poly(A) mRNA or 0.01 pg – 500 ng of specific RNA hexamer primer
25 µL 120 µL
100 µM IMPORTANT NOTES or gene-specific 2-20 pmol
transcript to generate first strand cDNA as the initial
Water, nuclease-free 1.25 mL 2 × 1.25 mL
Avoiding ribonuclease contamination step of a two-step RT-PCR protocol. primer
RNA purity and integrity are essential for synthesis of full- • Use 1 µg of isolated mRNA to generate first strand 10 mM dNTP Mix 1 µL (0.5 mM final
length cDNA. RNA can be degraded by RNase A, which is cDNA for second-strand synthesis and subsequent concentration)
CERTIFICATE OF ANALYSIS a highly stable contaminant found in any laboratory cloning reactions.
environment. To prevent contamination both the laboratory Water, nuclease-free to 15 µL
RT-PCR using 100 fg of control GAPDH RNA and environment and all prepared solutions must be free of Primers
GAPDH control primers generated a prominent 496 bp Synthesis of first strand cDNA can be primed with either 2. Optional. If the RNA template is GC-rich or is known
RNases.
product on 1% agarose gel after ethidium bromide oligo(dT)18 primer, random primers or gene-specific to contain secondary structures, mix gently,
General recommendations to avoid RNase contamination:
staining. primers. centrifuge briefly and incubate at 65 °C for 5 min.
• Use certified nuclease-free labware or DEPC-treat all Chill on ice, briefly centrifuge again and place on ice.
tubes and pipette tips to be used in cDNA synthesis. Oligo(dT)18 primes cDNA synthesis from the poly(A) tail
. present at the 3’-end of eukaryotic mRNA. Random 3. Add the following components in the indicated order:
• Wear gloves when handling RNA and all reagents,
Quality authorized by: Jurgita Zilinskiene primers initiate cDNA synthesis from the total RNA
as skin is a common source of RNases. Change 5X RT Buffer 4 µL
population (rRNA and mRNA). Therefore, using random
gloves frequently.
primers for first strand synthesis results in a greater Maxima H Minus Enzyme 1 µL
• Use RNase-free reagents, including high quality Mix
complexity of the generated cDNA compared with the
water (e.g., Water, nuclease-free, #R0581).
oligo(dT)18 primer. As a consequence, the sensitivity and Total volume 20 µL
• Keep the kit components tightly sealed when not in specificity of subsequent PCR reactions may be reduced.
use. Keep all tubes tightly closed during the reverse However, there are several applications where it is Mix gently and centrifuge. Incubate:– if an oligo(dT)18
transcription reaction. primer or gene-specific primer is used, incubate for 30 min
beneficial to use random primers, such as cDNA synthesis
using mRNAs without a poly(A) tail, or cDNA synthesis at 50 °C.
Rev.8 V using poly(A)-enriched RNA samples. – if a random hexamer primer is used, incubate for
10 min at 25 °C followed by 30 min at 50 °C.
Gene-specific primers are used to synthesize specific
cDNA from a pool of total RNA or mRNA and must be – for transcription of GC-rich RNA, the reaction
obtained by the user. temperature can be increased to 65 °C.
4. Terminate the reaction by heating at 85°C for 3. Add the following components to the reaction tube in TROUBLESHOOTING
5 minutes. the indicated order: Low yield or no product in RT-PCR or RT-qPCR
The reaction product of the first strand cDNA synthesis 5X RT Buffer 4 µL Poor integrity of RNA template.
can be used directly in PCR, or stored at -20 °C for up to RNA purity and integrity is essential for synthesis and
Maxima H Minus 1 µL quantification of cDNA. Always assess the integrity of
one week. For longer storage, -70 °C is recommended. Enzyme Mix RNA prior to cDNA synthesis Use freshly prepared RNA.
Avoid freeze/thaw cycles of the cDNA.
Total volume 20 µL Multiple freeze/thaw cycles of the RNA sample and
II. PCR synthesized cDNA is not recommended.
Mix gently and centrifuge. Follow general recommendations to avoid RNase
The product of the first strand cDNA synthesis reaction 4. Incubate for 10 min at 25°C followed by 15 min at 50 contamination and discard low quality RNA.
can be used directly in PCR. The volume of the first strand °C. Low template purity (inhibitors in RNA sample).
cDNA synthesis reaction mixture should not comprise Trace amounts of agents used in RNA purification
more than 1/10 of the total PCR reaction volume. 5. Terminate the reaction by heating at 85 °C for
5 minutes. protocols may remain in solution and inhibit first strand
Normally, 2 µL of the first strand cDNA synthesis reaction synthesis, e.g., SDS, EDTA, guanidine salts, phosphate,
mixture is used as template for subsequent PCR in a 25 Note. For RNA template quantities greater than 1 µg, pyrophosphate, polyamines, spermidine. To remove trace
µL total volume. Taq DNA polymerase, PCR Master Mix prolong the reaction time to 30 min. For RNA templates contaminants, re-precipitate the RNA with ethanol and
(2X) or Maxima Hot Start PCR Master Mix (2X) can be that are GC-rich or have a large amount of secondary wash the pellet with 75% ethanol.
used to amplify fragments less than 3 kb. Thermo structure, the reaction temperature can be increased to 65 Insufficient template quantity.
Scientific™ DreamTaq™ DNA polymerase is suitable for °C. Increase the amount of RNA template in the first strand
amplification of longer fragments up to 6 kb. Thermo reaction to the recommended level. Following DNase I
Scientific™ Phusion™ High-Fidelity DNA Polymerases are The reverse transcription reaction product can be used treatment, terminate the reaction by heat inactivation of
recommended to generate amplicons up to 20 kb. directly in qPCR, or stored at -20 °C for up to one week. the enzyme in the presence of EDTA (to bind Mg2+).
For longer storage, -70 °C is recommended. Avoid GC rich template.
RT-qPCR freeze/thaw cycles of the cDNA. If the RNA template is GC rich or is known to contain
secondary structures, the temperature of the reverse
I. First Strand cDNA Synthesis II. qPCR transcription reaction can be increased up to 65 °C.
The following protocol is optimized to generate first-strand The product of the first strand cDNA synthesis reaction Excess amount of cDNA in qPCR/PCR.
cDNA for use in two-step RT-qPCR. can be used directly in qPCR. The volume of first strand Decrease amount of cDNA synthesis reaction in
After thawing, mix and briefly centrifuge the components of qPCR/PCR. The volume of the cDNA synthesis reaction
cDNA synthesis reaction mixture should not comprise
the kit. Store on ice. mixture should not exceed 10% of the final PCR reaction
more than 1/10 of the total PCR reaction volume. Normally, mixture.
1. Add the following reagents into a sterile, RNase free 2 µL of the RT mixture is used as template for subsequent
No product in long RT-PCR (>5 kb)
tube on ice in the indicated order: qPCR in 25 µL total volume. Maxima H Minus First Strand
cDNA Synthesis Kit is optimized for use with Thermo Suboptimal priming.
total RNA 1 pg - 5 µg Use oligo (dT)18 primer or gene specific primer.
Template Scientific™ Luminaris™ qPCR master mixes.
or poly(A) mRNA 0.1 pg – 500 ng If random primers are used, reduce the amount of
RNA
or specific RNA 0.01 pg - 500 ng References random primers to 25 pmol per 20 µL reaction
Primers oligo (dT)18 primer 1. Wiame, I., et al., Irreversible heat inactivation of DNaseI mixture.
0.25 µL (25 pmol)
and random without RNA degradation, BioTechniques, 29, 252-256, Note:
0.25 µL (25 pmol) RT-PCR product longer than expected
hexamer primer or 2000. This product or the use of this product is covered by US patent
2-20 pmol RNA template is contaminated with DNA.
gene-specific primer 2. Fleige, S., Pfaffl, M.W., RNA integrity and the effect on application US20110065606A1 and corresponding counterparts. The
10 mM dNTP Mix 1 µL the real-time qRT-PCR performance, Mol. Aspects Med., Amplification of genomic DNA containing introns. To purchase of this product includes a non-transferable license to use this
27, 126-139, 2006. avoid amplification of genomic DNA, design PCR primers product for the purchaser's internal research. All other commercial uses
Water, nuclease-free to 15 µL of this product, including without limitation product use for diagnostic
3. Nolan, T., et al., Quantification of mRNA using real-time on exon-exon boundaries or perform DNase I digestion
prior reverse transcription. purposes, resale of product in the original or any modified form or
2. Optional. If the RNA template is GC-rich or is known RT-PCR, Nat. Protoc., 1, 1559-1582, 2006. product use in providing commercial services require a separate
to contain secondary structures, mix gently, RT-PCR product in RT-minus control license. For further information on obtaining licenses please contact
centrifuge briefly and incubate at 65 °C for 5 min. RNA template is contaminated with DNA. [email protected]
Chill on ice, briefly centrifuge again and place on ice. The presence of a PCR product in the negative control
(RT-) reaction indicates that the reaction is contaminated PRODUCT USE LIMITATION
with DNA. To avoid amplification of genomic DNA, design This product is developed, designed and sold exclusively for research
PCR primers on exon-exon boundaries or perform DNase purposes and in vitro use only. The product was not tested for use in
I digestion prior reverse transcription. diagnostics or for drug development, nor is it suitable for administration
to humans or animals.
Please refer to www.thermoscientific.com/onebio for Material Safety
Data Sheet of the product.

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