Invitrogen™ Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR, compatible with PAXgene™ Blood RNA Tubes

Catalog No.	Quantity
44-490-82	200 Preps

Catalog No. 44-490-82 Supplier Invitrogen™ Supplier No. 4449082

Description

Includes

Box 1

• Digestion Solution 20mL
• PAXgene Wash 300mL.

Box 2

• Stop Solution 2mL
• DNase 1 200 μL

The Stabilized Blood-to-C_T Nucleic Acid Preparation Kit for qPCR makes it possible to perform expression analysis directly from a 500μL aliquot of human whole blood collected in PAXgene Blood RNA tubes without conventional RNA purification.

The Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR makes it possible to perform expression analysis directly from a 500 μL aliquot of human whole blood collected in PAXgene™ Blood RNA tubes without conventional RNA purification. This simple procedure creates a lysate that can go directly into reverse transcription and real-time PCR with the sensitivity and specificity equivalent to conventionally purified RNA. This kit saves time, generates much less waste, and offers a simple workflow that is suitable for a few samples or can easily be incorporated into automated, high throughput applications.

• Detect total RNA (including miRNA) from PAXgene™ Blood RNA Tubes without purification
• Fast—Prepare 96 samples for TaqMan™-based real-time RT-PCR in under 1 hour
• Robust—500 μl sample processed at room temperature with results equivalent to those from purified RNA
• Flexible Format—Samples can be prepared in microfuge tubes or manual/automated 96-well plates.

Simple Workflow Without Sacrificing Sensitivity & Specificity

The process consists of pelleting and washing a 500uL aliquot from the blood tube, followed by a unique digestion/DNase step which preserves RNA integrity. The preparation takes place entirely at room temperature unlike other products that require temperature-specific incubation steps. Up to 96 samples can be prepared in less than an hour, half the time it takes for competitive spin column purification to process only 12 samples, with less hands-on effort. The resulting lysate can go directly into one-step or two-step qRT-PCR reactions or stored for later use (1 hour at 4°C or 5 months frozen). This straightforward procedure takes place in the same sample processing well, from start to finish, meaning no sample transfers. The real-time RT-PCR data generated from the lysate is equivalent to results obtained from conventionally purified RNA from the same tubes and has been validated with the following qPCR reagents:

• Applied Biosystems' High Capacity RNA-to-cDNA™ Kit and TaqMan™ Gene Expression Master Mix
• Invitrogen's SuperScript™ VILO™ cDNA Synthesis Kit and TaqMan™ Gene Expression Master Mix
• Applied Biosystems' TaqMan™ MicroRNA Reverse Transcription Kit and TaqMan™ Universal Master Mix II
• Applied Biosystems' TaqMan™ One-Step RT-PCR Master Mix Reagents Kit

Reverse transcription reagents for cDNA synthesis, TaqMan™ master mixes, and TaqMan™ assays for real-time PCR analysis are sold separately and are not included with the kit.

The total number of reverse transcription and PCR reactions possible from one starting sample depends on the minimum sample requirements of the desired RT-PCR kits. Please see the user documentation for more details.

Screen, Discover, or Validate

For users with specific genes of interest, the Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR was designed to enable quick screening from a small amount of starting sample (approximately 5% of the total volume of a properly filled PAXgene™ Blood RNA Tube per sample). The remainder of the blood tube can be archived for later use or purified as needed.

Those who perform discovery or validation experiments with real-time RT-PCR analysis will save effort, time, and overall cost with the Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR – while employing a scalable process allowing labs bottlenecked by conventional purification to finally start processing samples with ease.

miRNA Analysis

With the growing focus on non-coding RNA and its regulatory role in development and disease, it's reassuring to know these small but important RNA molecules can be detected properly with the Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR. Our scientists have tested the lysate with the Applied Biosystems TaqMan™ MicroRNA Reverse Transcription Kit and TaqMan™ Universal Master Mix II against a large panel of TaqMan™ microRNA assays and generated results equal to or better than those of the leading competitor's miRNA purification product.

Reduced Footprint & Less Waste = More Savings

Competitive products, especially column-based methods, generate considerable amounts of plastic and biohazardous waste per sample (spin columns, tips, microfuge tubes, and reagents). The Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR simplifies the workflow so the total plastic waste is 75% less by weight for a comparable number of samples processed via columns. The kit generates no organic waste. It also has a space-saving footprint to maximize freezer capacity. Reduced hazardous waste disposal and less plastic consumption translate into cost savings – as well as reducing the environmental impact.

For Users Needing Full Purification: If conventional RNA purification is still required for the remnant blood tube, one option is the MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit. This product maximizes your yield of total RNA (including miRNA) from PAXgene™ Blood RNA Tubes using proven magnetic bead-based separation technology.

One-Stop Shopping: The Stabilized Blood-to-C_T™ Nucleic Acid Preparation Kit for qPCR is compatible with our suite of products (including RT and PCR master mixes that must be purchased separately). Simplify the ordering process and reduce freight cost by getting more from one supplier.

Specifications:

• Compatibility— PAXgene™ Blood RNA Tubes
• Processing Time—Under 1 hour
• Format— microfuge tubes, 96-well plates
• Equipment Needed—Standard centrifuge capable of 5,000 x g with either 96-well plate or microfuge tube adaptors and 96-well plate or microfuge tube vortexer
• Number of Preps —200 reactions

NOTE: Blood tubes, reverse transcription reagents for cDNA synthesis, and TaqMan™ master mixes for real-time PCR analysis are sold separately.

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

PAXgene™ and PreAnalytiX™ are registered trademarks of PreAnalytiX GmbH. No sponsorship, endorsement, or affiliation is implied herein.

Specifications

• Column Type: Spin Column
• Content And Storage:

  Kit Components:
  Box 1-Store at 4°C
  • Digestion Solution, 20 mL
  • PAXgene™ Wash, 300 mL
  
  Box 2-Store at -20°C
  • Stop Solution, 2 x 1 mL
  • DNase 1, 200 μL

• Format: Kit
• Sample Type: Blood
• Final Product Type: Lysate
• For Use With (Application): Real-Time Quantitative PCR (qPCR)
• High-throughput Compatibility: High-throughput Compatible, Not High-throughput Compatible (Manual), Automated Protocols
• PCR Method: 1-step qRT-PCR, 2-step qRT-PCR
• Product Line: Ambion, Blood-to-CT
• Product Type: Nucleic Acid Preparation Kit
• Quantity: 200 Preps
• Target: Lysate, ready for real-time PCR
• Test Time: < 1 hr.

Frequently Asked Questions (FAQs)

What can I do to improve the sensitivity of my qPCR assay?

If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:

- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)

How do I set the baseline for my qPCR experiment?

Most times your instrument software can automatically set a proper baseline for your data. Check out our short video, Understanding Baselines, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=5BjFAJHW-bE).

How do I set the threshold for my qPCR experiment?

In most cases your instrument software can automatically set a proper threshold for your data. Check out our short video, Understanding Thresholds, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=H_xsuRQIM9M).

I am not getting any amplification with my TaqMan Assay or SYBR Green primer set. What is causing this?

There could be several reasons for no amplification from an assay or primer set. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/no-amplification.html) for more details.

I am getting amplification in my no-template control (NTC) wells in my qPCR experiment. What is causing this?

There could be several reasons for amplification in a NTC well. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/amplification-no-template-control.html) for more details.

My amplification curves have a funny shape in my qPCR experiment. What is causing this?

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html) for more details.

What can I do if the amplification of my target gene is later than expected for my qPCR experiment?

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool for more details (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html).

Can I use my SYBR Green primers for a TaqMan assay?

It may be possible to use your SYBR Green primers for a TaqMan assay, depending on how they were designed. You would have to design a separate probe to use with your existing primers. Please refer to the guidelines in this manual (https://tools.thermofisher.com/content/sfs/manuals/cms_041902.pdf) on “Manually Designing Primers and Probes” for the next steps. If you have Primer Express Software, you can use that software to design a probe. Please note that restricting the design using the predesigned SYBR primers may not allow for a successful probe design.

Do I have to normalize my samples for comparative Ct experiments?

Comparative Ct experiments use an endogenous control gene to normalize the cDNA input. Please watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=jst-3hD_xFQ) for more details on how this works. For a protocol workflow, please refer to our Guide to Performing Relative Quantitation of Gene Expression (https://tools.thermofisher.com/content/sfs/manuals/cms_042380.pdf).

What are the requirements for a relative quantification qPCR experiment?

In a relative quantification experiment, you will need to identify an endogenous control and a reference (or calibrator) sample. An endogenous control is a gene that does not change in expression across all the samples in your study. A reference sample is the sample that you are comparing all others to. This is often the untreated, or control, sample. Please see our Relative Gene Expression Workflow bulletin (https://tools.thermofisher.com/content/sfs/brochures/cms_075428.pdf) for more step-by-step guidelines on how to design your experiment.

What are the requirements for a standard curve qPCR experiment?

In a standard curve experiment, you must generate a standard curve for each target gene. The standards should closely represent the sample (i.e., RNA for RNA input, plasmid or gDNA for DNA input). This reference (http://www.ncbi.nlm.nih.gov/pubmed/11013345) is a good review of standard curves and the experimental setup. You can also review this short video (https://www.youtube.com/watch?v=mE5ieko9_RQ) on standard curve experiments.

What is the difference between absolute quantification (AQ) and relative quantification (RQ)? How do I choose which method to use?

Absolute quantification will quantitate unknowns based on a known quantity. It involves the creation of a standard curve from a target of known quantity (i.e., copy number). Unknowns can then be compared to the standard curve and a value can be extrapolated. Absolute quantification is useful for quantitating copy number of a certain target in DNA or RNA samples. The result usually is a number followed by a unit, such as copy number and ng, etc.

Relative quantification can quantitate a fold difference between samples. It involves the comparison of one sample to another sample (calibrator) of significance. For example, in a drug treatment study you could compare a treated to an untreated sample. The quantity of the calibrator is not known and cannot be measured absolutely. Therefore the calibrator (untreated sample) and samples (treated samples) are normalized to an endogenous control (a gene that is consistently expressed among the samples) and then compared to each other to get a fold difference. Relative quantification is useful for quantitating messenger RNA levels. Since the result is a fold change or ratio, it is not followed by a unit.

The method that you choose will depend on the type of data you need from your experiment. You can find more information here (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html) as well.

Can I do a melt curve with a TaqMan assay?

No. A TaqMan probe, once cleaved, cannot be re-quenched. Therefore a melt curve does not apply when using a TaqMan assay.

What is the difference between TaqMan and SYBR Green methods of detection?

TaqMan and SYBR Green chemistries are two different methods of detection for qPCR. Please see this detailed comparison of these two approaches (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/taqman-assays-vs-sybr-green-dye-for-qpcr.html). You can also watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=fkUDu042xic) on how TaqMan assays work.

How many replicates do I need to run for my qPCR experiment? What recommendations do you have for plate setup?

Please view this short video (https://www.youtube.com/watch?v=eIaPGhOjBQo), which explains some best practices for replicates and plate setup.

What are the different phases of a qPCR reaction?

Check out this short video (https://www.youtube.com/watch?feature=player_embedded&v=4sXPUbIrh3A) to understand the different phases of the PCR reaction and why they are important.

I'm trying to decide between purchasing a one-step or two-step RT-PCR kit. Can you review the advantages and disadvantages of each?

One-step RT-PCR is convenient and less prone to contamination, as there is less opportunity for pipetting error. This method is also faster than the two-step process. However, the cDNA cannot be archived, and fewer genes can be analyzed. Two-step RT-PCR gives you the ability to archive cDNA, analyze multiple genes, and offers greater flexibility. Learn more about the difference between one-step and two-step RT-PCR on this page Onestep vs Twostep RT-PCR.

Have you validated the Stabilized Blood-to-CT Nucleic Acid Preparation Kit for qPCR, compatible with PAXgene Blood RNA Tubes (Cat. No. 4449082) with the TaqMan Advanced miRNA Assays?

We have not tested this kit with the TaqMan Advanced miRNA Assay. We have also not tested blood as sample to use directly with the TaqMan Advanced miRNA cDNA Synthesis Kit. Blood usually has a lot more impurities compared to serum or plasma, so it would be best to extract RNA from blood followed by the TaqMan Advanced cDNA synthesis reaction.

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