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Crescendo cDNA™ Synthesis for qPCR

Robust solution for enhanced qPCR detection from low input and degraded samples




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Tab 01 / Overview
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Crescendo cDNA Synthesis for qPCR - High-Sensitivity for low input and degraded samples

The Crescendo cDNA™ Synthesis for qPCR kit provides a simple, robust method to prepare amplified cDNA from total RNA samples.

When starting with low input and degraded RNA samples, current cDNA synthesis methods such as standard first-strand synthesis, can require excessive PCR cycles or provide false negative results. This can have serious consequences especially for applications such as SARS-CoV-2 detection from nasal swab samples.

Crescendo has been designed to provide improved sensitivity over the standard single strand cDNA synthesis method. Utilizing Tecan’s user-friendly SPIA (Single Primer Isothermal Amplification) technology, Crescendo cDNA synthesis generates more copies of each transcript than other library prep techniques making it ideal for the detection of low abundance transcripts.

The amplified cDNA can provide increased sensitivity of detection during qPCR without modifying relative gene expression and is ideal for gene expression analysis using real-time qPCR, TaqMan® arrays or sample archiving.

The Crescendo cDNA Synthesis for qPCR kit features:

  • Broad input range of 500 pg to 50 ng of total RNA
  • Simple workflow completed in 4 hours
  • Compatible with degraded RNA samples
  • Whole transcriptome cDNA conversion and amplification
  • Increased detection sensitivity for low-abundant transcripts even with compromised samples

Applications

  • Quantitative real-time PCR
  • TaqMan array
  • Sample archiving




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Tab 02 / Highlights
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Compatible with low input, compromised nasal swab samples

  • Convert and amplify cDNA even from highly degraded RNA isolated from nasal swabs
  • Generate hundreds of nanograms of cDNA for multiple studies or archiving
  • Improved detection sensitivity compared to standard first strand synthesis

Crescendo amplified cDNA provides increased sensitivity of detection of SARS-CoV-2 from nasal swabs. Total RNA was isolated from a COVID-19 positive nasal swab. The resulting RNA had low yield (2.8 ng/µL) and was significantly degraded with a RIN of 2.3. Approximately 7 ng of total RNA was used as input into the Crescendo cDNA Synthesis for qPCR kit or a first strand synthesis kit. SARS-CoV-2 was detected in the Crescendo cDNA with the N1 and N2 (shown) probes but was not detected with the first strand synthesis only workflow, even after 45 cycles. The cDNA conversion and amplification provided by the Crescendo cDNA Synthesis for qPCR kit provides a significant advantage in detection sensitivity for low input and degraded samples reducing inconclusive results.

Increase your sensitivity of SARS-CoV-2 detection

  • Increased detection sensitivity compared to standard first strand cDNA synthesis
  • Accurate quantification with minimum change to gene expression levels
  • Detect your target gene earlier in the qPCR assay
  • Avoid non-specific background with fewer PCR cycles

Crescendo amplified cDNA provides better detection of SARS-CoV-2. A 10-fold dilution series of SARS-CoV-2 RNA, ranging from 250,000 to 250 copies was added to a background of 2.5 ng of K562 total RNA. The RNA was converted to cDNA using a standard first strand synthesis kit or the Crescendo cDNA Synthesis for qPCR kit. The CDC SARS-CoV-2_N2 TaqMan assay showed that SARS-CoV-2 was detected significantly earlier with the Crescendo amplified cDNA compared to the first strand synthesis method which required significantly more PCR cycles at the low input ranges.

Robust cDNA conversion and amplification

  • Whole transcriptome cDNA conversion and amplification allows you to assay any target gene
  • Generate sufficient yield for all your experimental needs even from low input and poor-quality RNA
  • Conversion of RNA samples to cDNA eliminates worry about storage of unstable RNA

 

Across a broad range of high-quality total RNA inputs, Crescendo generates over a microgram of cDNA. Even with 500 pg of total RNA, data shows that Crescendo provides sufficient amounts of cDNA for all your qPCR and archiving needs.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

All major qPCR machines

Amplification Type

Amplification Type

Whole transcriptome

Starting Material

Starting Material

Total RNA

Input Amounts

Input Amounts

500 pg – 50 ng




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Tab 04 / Faq
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FAQ

What materials are provided with the Crescendo cDNA Synthesis for qPCR kit?

The Crescendo cDNA Synthesis for qPCR kit provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification, yielding cDNA. The kit provides nuclease-free water but Agencourt beads must be purchased separately. A detailed list of required equipment and reagents is provided in the user guide.

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer and a thermal cycler. A UV/Vis spectrophotometer and an Agilent Bioanalyzer will be useful.

What additional consumables does the user need?

A detailed list of equipment, reagents and labware that are required for use of the Crescendo cDNA Synthesis for qPCR kit is provided in the user guide.

Is the Crescendo cDNA Synthesis for qPCR kit 3´ biased?

No, first strand cDNA is primed with random hexamers as well as an oligo(T) primer providing whole transcriptome cDNA conversion and amplification.

Has Tecan performed reproducibility studies on the Crescendo cDNA Synthesis for qPCR kit?

Yes. Sample-to-sample, lot-to-lot and operator-to-operator reproducibility studies are routinely conducted.

the Crescendo cDNA Synthesis for qPCR kit for archiving cDNA?

Amplified cDNA may be stored at -20°C for at least several months.

Can I use TRIzol® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol® or similar methods as any carry over of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Technical Support for more information.

Do I need to use high quality total RNA?

Yes. Use of lower quality RNA may result in poor performance. One approach to determining RNA quality is the Agilent Bioanalyzer’s RNA Integrity Number (RIN). Clean RNA with a RIN score greater than 7 should amplify well.

How much total RNA do I need for amplification?

We recommend staying within the range of 500 pg to 50 ng total RNA starting material. Amounts greater than 50 ng may produce variable results.

Can the Crescendo cDNA Synthesis for qPCR kit amplify DNA?

The Crescendo cDNA Synthesis for qPCR kit is designed to amplify RNA, not DNA.

Can I use the Crescendo cDNA Synthesis for qPCR kit on bacterial RNA samples?

The Crescendo cDNA Synthesis for qPCR kit theoretically will work with some bacterial RNAs. However, the kit has not been optimized for this purpose.

Are there any tissues that will not work with the Crescendo cDNA Synthesis for qPCR kit?

We have not encountered any good quality, clean RNA samples that will not work with the Crescendo cDNA Synthesis for qPCR kit.

Can I perform fewer than 8 reactions at a time?

We recommend a minimum batch size of 8 reactions. Smaller batch sizes may result in difficulty pipetting small volumes and lead to poor performance.

Does the Crescendo cDNA Synthesis for qPCR kit generate product in the absence of RNA input?

Due to the high sensitivity inherent in our amplification systems a non-specific product can be generated in the absence of input RNA. However, in the presence of even a very small amount of RNA the amplified cDNA has been demonstrated to be specific. To minimize the potential for carryover of non-specific amplification products into future reactions, we recommend using a low-input (i.e. 50-100 pg) rather than a no-input (i.e. negative) control.

How many rounds of amplification are performed with the Crescendo cDNA Synthesis for qPCR kit?

The Crescendo cDNA Synthesis for qPCR kit performs a single round of amplification.

Do I need to order specific primers for the amplification?

No. The DNA/RNA primers provided in the Crescendo cDNA Synthesis for qPCR kit are universal.

Do I have to use the DNA/RNA primers supplied with the kit?

Yes. The Crescendo cDNA Synthesis for qPCR kit will not work properly with other primers.

If I choose to purify my product, what method do you recommend?

Several purification options are available for the final SPIA cDNA cleanup step. These are described in Appendix B of the User Guide. Selection of the optimum purification option can depend on many factors. Please contact the Tecan Technical Support team for assistance in selecting the appropriate option for your application. Refer to section II.B for ordering information.

Where can I safely stop in the protocol?

We do not recommend stopping at any stage of the protocol.

How much cDNA can I expect from a single reaction?

We expect close to a microgram of cDNA when starting with 500 pg of high quality total RNA. The total yield can vary depending on the RNA quantity and quality.

Is the cDNA yield dependent upon the quantity of total RNA input?

Yes, higher RNA inputs will produce higher yields. However, at inputs of above 50 ng, the yields may become variable without increasing.

What size cDNA is generated by the Crescendo cDNA Synthesis for qPCR kit?

The Crescendo cDNA Synthesis for qPCR kit generates cDNA ranging in size from 200 bp to 1.5 kb for high quality RNA.

Should I purify the cDNA before determining the concentration?

Yes. The primers and reagents present in the amplified cDNA will interfere with accurate quantitation. Details on measuring the concentration of cDNA are included in the User Guide.

Where in my target sequence can I design my qPCR primers?

The Crescendo cDNA Synthesis for qPCR kit provides whole transcript conversion and amplification. Therefore, primers can be designed at any location within the transcript. We recommend designing amplicons between 50 to 200 bases. For degraded RNA samples, smaller amplicons may provide better results.

How many qPCR reactions will I get from one amplification reaction?

The number of qPCR reactions depends on the abundance level of the genes being interrogated. For medium to high copy genes, the cDNA may be diluted as much as 400-fold, enough for hundreds of qPCR reactions. For very low copy genes you will need to use more cDNA per reaction. You will need to determine how much cDNA to use per reaction depending on the abundance of the gene being interrogated.




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Tab 05 / Literature
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Literature

Type
Title
No
Product Sheet
401798 V1
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For research use only. Not for use in diagnostic procedures.