TaqMan™ RNase P Detection Reagents Kit - FAQs

With the Oncomine Focus Assay, Chef-Ready Library kit, where can I find the combined barcoded AmpliSeq libraries when the run is complete?

After the completion of a run, the Ion Chef Instrument holds the barcoded libraries in the tube loaded in Position D of the Reagents cartridge. To avoid fluid loss due to evaporation, remove and cap the tube of combined barcoded libraries as soon as possible after run completion. Do not leave the tube in the instrument longer than 24 hours after the start of the run. After 24 hours from the start of the run, the instrument chiller will stop actively cooling, and the sample will be held at 27 degrees C.

The tube in Position D of the Ion AmpliSeq Chef Reagents DL8 Cartridge will contain 700 µL of combined barcoded libraries at completion of the run. The libraries are at >>100 pM (total combined library concentration) and are ready to use in template preparation. One can store unused portions of combined libraries at 4 to 8 degrees C for up to 1 month. For longer-term storage, store at -30 to -10 degrees C.

With the Oncomine Focus Assay, Chef-Ready Library kit, can I process fewer than 8 samples per Ion AmpliSeq run on the Ion Chef System?

Yes, fewer than 8 samples may be processed per run but keep in mind that a run consumes kit reagents for 8 samples regardless of the sample number.

If I process less than 8 samples using the Oncomine Focus Assay, Chef-Ready Library kit, do I need to quantitate the final combined output library?

If you are processing ≤5 samples, we recommend that you quantify your output combined library by qPCR to ensure that an optimal concentration is used in templating reactions.

If you are processing fewer than 8 samples, it is preferable to add replicates or positive control samples to the run. Otherwise, pipet 15 µL of nuclease-free water as non-template control into column 1 wells that do not contain a DNA sample.

What is the RNA input for the Oncomine Focus Assay, Chef-Ready Library kit?

Each reverse transcription reaction requires 10 ng of DNase-treated total RNA (≥1.43 ng/µL).

What is the DNA input for the Oncomine Focus Assay, Chef-Ready Library kit?

For each target amplification reaction, use 300-30,000 copies of DNA (10 ng of mammalian gDNA) from normal or FFPE tissue.

With the Oncomine BRCA Research Assay, which method do you recommend for quantifying gDNA?

We recommend two methods for gDNA quantification:

- Qubit 3.0 Fluorometer (Cat. No. Q33216) with Qubit dsDNA HS Assay Kit (Cat. Nos. Q32851, Q32854)
- qPCR using TaqMan RNase P Detection Reagents Kit (Cat. No. 4316831)

What is the purpose of the RNase P Detection Reagents Kit (Cat. No. 4316831), and what is provided in the kit?

RNase P is a single-copy gene encoding the RNA moiety for the RNase P enzyme, and it is an excellent gene to use as a reference/control for Copy Number Variation (CNV) assays. The TaqMan RNase P Detection Reagents Kit contains a 20X mix of primers and probe (FAM dye-labeled) that will detect and quantitate genomic copies of the human RNase P gene. The primers and probe are designed according to TaqMan chemistry guidelines for quantitation, and utilize the universal thermal cycling parameters. This kit is used to perform a 5' nuclease assay with TaqMan Universal PCR Master Mix (Cat. No. 4304437) or the TaqMan Core PCR Reagents Kit (Cat. No. N808-0228) with genomic, plasmid or complementary DNA (cDNA) templates.

The complete components of the TaqMan RNase P Detection Reagents Kit, Cat. No. 4316831, are: 20X RNase P Primer-Probe (FAM/TAMRA probe) Mix, 1 tube Human Genomic Control DNA, 10 ng/ul (100 ul), and the Product Insert with an overview and procedure for the use of the kit.