Crescendo cDNA™ Synthesis for qPCR

Product Details

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

Quantitative PCR (qPCR) is a sensitive method that allows researchers to detect and quantify nucleic acids in a starting material. For RT-qPCR studies, however, the RNA input, quality and expression of the target have a significant impact on the ability to detect the gene of interest. Standard first strand synthesis, when starting with low input and degraded RNA samples, 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.

The Crescendo cDNA™ Synthesis for qPCR kit provides a simple, robust method to prepare amplified cDNA from total RNA samples. Amplified cDNA increases the number of cDNA copies which provides improved sensitivity of detection during qPCR studies. Whole transcriptome cDNA conversion and amplification with the Crescendo cDNA Synthesis kit is powered by SPIA^® (Single Primer Isothermal Amplification) technology, a simple, sensitive, highly published process for unbiased cDNA amplification developed by Tecan. Crescendo cDNA Synthesis kit provides efficient conversion and amplification across a range of RNA inputs and quality. 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

Back to Microarrays and qPCR

Product Highlights

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

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

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

Applications

Quantitative real-time PCR
TaqMan array
Sample archiving

Specifications

Specification	Description
Compatible Platform	All major qPCR machines
Amplification Type	Whole transcriptome
Starting Material	Total RNA
Input Amounts	500 pg – 50 ng

Resources

FAQs

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Getting started

What materials are provided with the Ovation qPCR System?

The Ovation qPCR System provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification, yielding cDNA. The kit also provides nuclease-free water.

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?

None. For the optional purification of amplified cDNA, see user guide for validated purification products and procedures.

Is the Ovation qPCR System 3´ biased?

No, this product is different from the Ovation RNA Amplification System in that the first strand cDNA is primed with random hexamers as well as a 3´ primer.

What is the dynamic range of input mRNA that is amplified with the Ovation qPCR System?

Our studies demonstrate amplification over 6 orders of magnitude starting with transcripts present as low as 20 copies in a sample.

Has NuGEN performed reproducibility studies on the Ovation qPCR System?

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

Can I use the Ovation qPCR System for archiving cDNA?

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

Input recommendations

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

Norgen Biotek Total RNA Purification Kit
Zymo Research Quick-RNA™ Kits
Arcturus PicoPure^® RNA Isolation Kit
Ambion PureLink^® RNA Mini Kit
Qiagen RNeasy Kits Organic methods such as TRIzol^® Reagent should be subsequently followed with a column-based clean-up method.

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 5 to 50 ng total RNA starting material. Amounts greater than 50 ng may produce variable results.

Can the Ovation qPCR System amplify DNA?

The Ovation qPCR System is designed to amplify mRNA, not DNA.

Can I use the Ovation qPCR System on bacterial RNA samples?

The Ovation qPCR System 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 Ovation qPCR System?

We have not encountered any good quality, clean RNA samples containing poly(A)+ RNA that will not work with the Ovation qPCR System.

General workflow

Can I perform fewer than 8 reactions at a time?

We recommend 3 batches of 8 reactions. Smaller batch sizes may result in fewer than 24 reactions in total.

Does the Ovation qPCR System 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 Ovation qPCR System?

The Ovation qPCR System performs a single round of amplification.

Do I need to order specific primers for the amplification?

No. The DNA/RNA primers provided in the Ovation qPCR System are universal.

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

Yes. The Ovation qPCR System 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 NuGEN 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.

cDNA quantification/qualification

What is the amplification efficiency of the Ovation qPCR System?

Based on qPCR on a variety of genes, an average amplification efficiency of 1500-fold is observed.

How much cDNA can I expect from a single reaction?

You should expect 1.5 to 4 μg of cDNA from 5 to 50 ng total RNA starting material.

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 Ovation qPCR System?

On a Bioanalyzer, using the RNA 6000 size markers, the average size of the amplified cDNA is 375 bases. More than 50% of the product is greater than 320 bases in length.

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.

Using amplified cDNA for qPCR

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

The Ovation qPCR System does not have a 3 prime bias and, therefore, primers can be designed at any location within the mRNA. In order to avoid qPCR interference from possible genomic DNA contamination, we recommend treating your RNA with DNase and designing your amplicons to span an intron.

How many qPCR reactions will I get from one Ovation qPCR System amplification?

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.

Ordering Information

Description	Part Number	No. of Reactions
Crescendo cDNA Synthesis for qPCR	30183903	16
Crescendo cDNA Synthesis for qPCR	30183905	64

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