Thermo Fisher Scientific RNA UltraSense One-Step Quantitative RT-PCR System User guide

Brand: Thermo Fisher Scientific

Model: RNA UltraSense One-Step Quantitative RT-PCR System

Type: User guide

RNA UltraSense™ One-Step Quantitative RT-PCR System

Catalog Numbers 11732927

Doc. Part No. 11732927.pps Pub. No. MAN0001007 Rev. 3.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermoﬁsher.com/support.

Product description

The RNA UltraSense™ One-Step Quantitative RT-PCR System is a one-step quantitative RT-PCR system for the sensitive, reproducible

detection of low-abundance RNA molecules using real-time detection instruments. This system combines the high-temperature reverse

transcription capability of SuperScript™ III Reverse Transcriptase with the automatic hot-start PCR provided by Platinum™ Taq DNA

Polymerase in a highly concentrated formulation that is optimized for the detection of viral RNA and rare transcripts. Both cDNA synthesis

and PCR are performed in a single tube using gene-specic primers and RNA. All components necessary for real-time RT-PCR are mixed

in the tube, and reverse transcription is directly followed by PCR cycling without additional handling, enabling sensitive detection from as

few as 10 copies of highly dilute RNA template.

Contents and storage

Contents Amount Storage

RNA UltraSense™ Enzyme Mix[1] 250 µL

15°C to 30°C[2]

RNA UltraSense™ 5X Reaction

Mix[3] 1 mL

20X Bovine Serum Albumin (BSA),

UltraPure, non-acetylated, 1

mg/mL

300 µL

Magnesium Sulfate (MgSO4), 50

nM 1 mL

ROX™ Reference Dye[4] 100 µL

[1] Includes RNaseOUT™ Recombinant Ribonuclease Inhibitor.

[2] Stabiity can be extended by storing at -80°C

[3] Contains 1 mM of each dNTP.

[4] Store in the dark.

Quality control

The RNA UltraSense™ One-Step Quantitative RT-PCR System is

tested functionally by quantitative real-time analysis using total

HeLa RNA as template. Kinetic analysis must demonstrate a linear

dose response with decreasing target concentration and

detection of β-actin mRNA in 1 pg of total HeLa RNA.

Required material not supplied

Unless otherwise indicated, all materials are available through

thermoﬁsher.com. "MLS" indicates that the material is available

from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

One of the following instruments, or equivalent[1]

Applied Biosystems™ 7500 Fast Food Safety

Real-Time PCR Instrument

A30304 (desktop)

A30299 (laptop)

Contact your local

microbiology sales

representative

QuantStudio™ 5 Food Safety Real-Time PCR

Instrument

A36320 (desktop)

A36328 (laptop)

Contact your local

microbiology sales

representative

Additional materials for PCR

Vortex mixer

Available through the

Thermo Fisher

Microbiology ordering

process. See

thermoﬁsher.com/

plastics for more

information.

8-channel pipette, 10‑ to 100‑µL

Filtered pipette tips, 10‑ to 100‑µL

Benchtop microcentrifuge with adaptors for

PCR plates and/or tubes

1.5-mL nuclease-free microcentrifuge tubes

Powder-free disposable gloves

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

Item Source

Optical reaction plates and covers, or optical PCR tubes and

caps

MicroAmp™ Fast Optical 96-Well Reaction

Plate, 0.1 mL 4346907

MicroAmp™ Optical Adhesive Film, 100 covers 4311971

MicroAmp™ Fast 8-Tube Strip, 0.1 mL

(See below for caps.) 4358293

MicroAmp™ Optical 8-Cap Strips 4323032

Reagents

Nuclease-free Water (not DEPC-Treated) AM9938

Ethanol MLS

[1] Other Applied Biosystems™ 7500 Fast Real‑Time PCR Instrument and

QuantStudio™ 5 Real‑Time PCR Instrument are also suitable.

Procedural guidelines

Guidelines for reaction setup and conditions

• RNA should be devoid of any RNase contamination and

aseptic conditions should be maintained. RNaseOUT™

Recombinant Ribonuclease Inhibitor is included in the

enzyme mix to safeguard against degradation of target RNA

due to ribonuclease contamination of the RNA preparation.

• The RNA UltraSense™ 5X Reaction Mix includes MgSO4 at a

concentration that facilitates primer binding with difcult

templates such as viral RNA. If necessary, use the 50-mM

MgSO4 solution included in the kit to increase the

magnesium concentration.

• Keep all components, reaction mixes and samples on ice.

After reaction assembly, transfer them to a thermal cycler

pre-heated to the desired cDNA synthesis temperature (45–

60°C) and immediately start the RT-PCR amplication

program.

•Efcient cDNA synthesis can be accomplished in a

incubation at 45–60°C for 15-30 minutes. Optimal

temperature varies for different primers and templates. A

good general starting point is 50°C for 15 minutes. For

problematic templates, or to increase the specicity of cDNA

priming, increase the cDNA synthesis temperature to 55°C.

• The SuperScript™ III Reverse Transcriptase is inactivated, the

RNA/cDNA hybrid is denatured, and the Platinum™ Taq DNA

Polymerase is activated during the incubation at 95°C for 2

minutes.

• The annealing temperature should be 0–10°C below the

melting temperature of the primers used.

• The extension time of 30 seconds for instruments that use

PCR tubes/plates is appropriate for the short amplicons that

are typically used in real-time PCR.

Guidelines for primer selection

•Gene-specic primers (GSP) are required. We do not

recommend using oligo(dT) or random primers, which may

generate nonspecic products in the one-step procedure

and reduce the amount of product.

• For mRNA, design primers that anneal to exons on both

sides of an intron or within the exon/exon boundary to allow

differentiation between amplication of cDNA and potential

contaminating genomic DNA.

• Primers should be designed according to standard PCR

guidelines. They should be specic for the target sequences,

be free of internal secondary structure, and should avoid

complementation at 3′ end within each primer, primer pair, or

hybridization probe sequence (except as required for hairpin

uorogenic primers such as LUX™ primers). For best results,

the amplicon size should be limited to 80–200 bp.

• Optimal results may require a primer titration between 100

and 500 nM. A nal concentration of 200 nM per primer is

effective for most reactions.

• For multiplex applications, limit the amount of primer for the

reference gene, such as β-actin or GAPDH, to avoid

competition between amplication of the reference gene and

sample gene. In general, the nal concentration of the

reference gene primer should be between 25 and 100 nM.

However, a primer titration is recommended for optimal

results.

Guidelines for dual-labeled probes

• The optimal concentration of probe may vary between 50

and 800 nM. A recommended starting concentration is 100

nM.

• The probe sequence should be free of secondary structure

and should not hybridize to itself or to primer 3′ ends.

• For multiplex applications, the concentration of each probe

may need to be adjusted independently to obtain optimal

uorescent signals. The amount of probe for the reference

gene, such as β- actin or GAPDH, should be limited as

described above for primers.

2RNA UltraSense™ One-Step Quantitative RT-PCR System User Guide

Set up the RT-PCR reactions

1. Set up the real-time PCR instrument as indicated in the following table

Table 1 Cycling conditions

Step No. of cycles Temp. Time

Reverse transcription 1 50°C[1] 15 min

Polymerase activation 1 95°C 2 min

Ampliﬁcation

40-50

95°C 15 sec

60°C 30 sec

Melt curve (optional) 1 Refer to the real-time PCR instrument doumentation.

[1] 42-50°C is acceptable. Use 50°C as a starting point.

Optimal temperatures and incubation times may vary for different target sequences.

2. On ice, prepare an RT-PCR Master Mix for the number of reactions required plus 10% overage.

Component 10-µL RT-PCR reaction 20-µL RT-PCR reaction

RNA UltraSense™ Enzyme Mix 0.5 µL 1 µL

RNA UltraSense™ 5X Reaction Mix 2 µL 4 µL

LUX™ Primers[1]

TaqMan™ Probes [2]

0.2 µL each 0.4 µL each

(Optional) ROX™0.2 µL 0.4 µL

Total Master Mix Volume 3.1 µL 6.2 µL

[1] Labeled primer: 10 µM, Unlabeled primer: 10 µM

[2] Primer pair: 10 µM each, Fluorogenic probe: 10 µM

3. On ice, add template and Nuclease-free Water (not DEPC-Treated) to the RT-PCR Master Mix according to the following table.

Component 10-µL RT-PCR reaction 20-µL RT-PCR reaction

RT-PCR Master Mix 3.1 µL 6.2 µL

Template Up to 6.9 µL Up to 13.8 µL

Nuclease-free Water (not DEPC-Treated) To 10 µL To 20 µL

Total RT-PCR Volume 10 µL 20 µL

4. Cap or seal the reaction vessels, and gently mix to make sure that all components are at the bottom of the amplication tube.

Centrifuge briey if needed.

5. Place reactions in a preheated thermal cycler programmed as described above. Collect data and analyze results. After cycling, hold

the reaction at 4°C until further analysis.

Troubleshooting

Observation Possible cause Recommended action

No ampliﬁcation product

Relative ﬂuorescent signal ≤

background or no template control

cDNA synthesis temperature too

high, low priming efﬁciency.

Lower incubation temperature.

RT or cDNA primer blocked by

secondary structure.

Raise incubation temperature. Redesign primer(s).

RNA has been damaged or

degraded.

Replace RNA if necessary.

RNA UltraSense™ One-Step Quantitative RT-PCR System User Guide 3

Observation Possible cause Recommended action

No ampliﬁcation product

Relative ﬂuorescent signal ≤

background or no template control

(continued)

RNase contamination. Maintain aseptic conditions; add RNase inhibitor.

Fluorescent probe not functional. Validate probe design and presence of ﬂuorophore and quencher:

Treat TaqMan™ probe with DNase, and check for increase in

ﬂuorescence. Redesign and/or resynthesize probe if necessary.

Poor sensitivity Not enough starting template RNA. Increase the concentration of template RNA; use 10 ng to 1 μg of

total RNA.

Product detected at higher than

expected cycle number

RNA has been damaged or

degraded.

Replace RNA if necessary.

RNase contamination. Maintain aseptic conditions; add RNase inhibitor.

RT inhibitors are present in RNA. Remove inhibitors in the RNA preparation by an additional 70%

ethanol wash. Inhibitors of RT include SDS, EDTA, guanidium salts,

formamide, sodium phosphate and spermidine.

Inefﬁcient cDNA synthesis. Adjust cDNA synthesis temperature and/or primer design.

Inefﬁcient PCR ampliﬁcation. Optimize PCR conditions:

• Adjust annealing temperature as necessary.

• Increase magnesium concentration.

• Redesign primers.

Higher than expected signal Too much sample added to

reactions.

Decrease the concentration of template RNA.

Product detected at lowerthan-

expected cycle number, and/or

positive signal from no-template

controls

Template or PCR carry-over

contamination.

Isolate source of contamination and replace reagent(s). Use separate

dedicated pipettors for reaction assembly and post-PCR analysis.

Assemble reactions (except for target addition) in a DNA-free area.

Use aerosol-resistant pipet tips or positive displacement pipettors.

Unexpected bands after

electrophoresis

RNA contamination with genomic

DNA.

Pre-treat RNA with DNase I.

Oligo(dT) or random primers used

for ﬁrst-strand synthesis.

Use gene-speciﬁc primers.

Low speciﬁcity in PCR. Optimize PCR conditions as described above.

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The information in this guide is subject to change without notice.

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PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

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1 June 2020

Thermo Fisher Scientific RNA UltraSense One-Step Quantitative RT-PCR System User guide

Thermo Fisher Scientific RNA UltraSense One-Step Quantitative RT-PCR System User guide

Thermo Fisher Scientific RNA UltraSense One-Step Quantitative RT-PCR System User guide

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