Performance:
The RT-qPCR kit ensures fast and easy preparation with a minimum of pipetting steps and is highly recommended for:

• direct detection of RNA viral pathogens in various tissues
• direct amplification of target RNA from sample materials
• point-of-care Diagnostics

Description:
The kit is recommended for use with Dual Labeled Fluorescent Probes, e.g. TaqMan®, Molecular Beacons or FRET probes but can also be used without fluorescent probes in standard PCR assays. The kit contains an enzyme mixture including a genetically engineered reverse transcriptase and an antibody-inhibited Taq polymerase. The 2x conc. reaction mix contains ultrapure dNTPs and an unique buffer system optimized to resist various PCR inhibitors in unpurified sample material

One.Direct.Step RT-qPCR for Probes is designed for quantitative real-time analysis of target RNA directly from whole blood, swabs and animal- or plant tissue without the requirement of any prior RNA purification steps

shipping and storage: transportation with blue ice; storage @ -20°C for at least 16 months (stable @ +4°C up to 4 weeks), avoid frequently freeze/thaw cycles

Manufactured and quality-controlled in accordance with ISO 9001:2000

Kit content:
Extraction Buffer: 10x concentrated
Direct Enzyme: Mix of engineered reverse transcriptase, antibody-inhibited hot start polymerase and RNase inhibitor in storage buffer with 50 % glycerol (v/v)

Direct Reaction Mix: 2x conc. buffer system containing dNTPs, enhancer and stabilizer
PBS (phosphate buffered saline): 10x concentrated 
PCR-grade Water

Preparation:

1. Whole Blood or Salvia (heparin-, EDTA- or citrate-treated whole blood)

• Add 1-5 _l of the sample without any pre-treatment directly to the RT-PCR assay.

2. Swab Samples

• Place the swab brush into a 1.5 ml microcentrifuge tube containing 270 µl PCR-grade Water and 30 µl PBS, 10x conc.

• Rotate the brush 5-10 times.

• Squeeze the brush and remove it from the tube.

• Centrifuge at 12,000 g for 3 min at room temperature.

• Discard the supernatant.

• Add 90 µl PCR-grade Water and 10 µl Extraction Buffer to the harvested sample.

• Briefly mix the sample by vortexing and make sure that the sample is soaked with Extraction Buffer.

• Incubate for 3 min at room temperature for tissue lysis and RNA releasing.

• Centrifuge briefly and transfer 1-5 _l of the supernatant to the RT-PCR assay.

• The lysate (supernatant) can be stored at -20°C for several weeks.

3. Animal or Plant Tissue

• Prepare a small piece from animal or plant tissue not exceeding 6 mm in diameter.

• Crack plant seeds to less than 1 mm in diameter using a BeadBeater, TissueLyser or small hammer.

• Add Extraction Buffer to the tissue sample as following:
 

Mix briefly by tapping or vortexing. Make sure that the sample is soaked with Extraction Buffer.

• Incubate for 3 min at room temperature for tissue lysis and RNA releasing.

• Centrifuge briefly and transfer 1-5 _l of the supernatant to the RT-PCR assay.

• The lysate (supernatant) can be stored at -20°C for several weeks.
 
Preparation of the RT-PCR Assay

Add the following components to a nuclease-free microtube. Pipette on ice and mix the components by pipetting gently up and down.
 

¹⁾  Direct Enzyme Mix already contains RNase inhibitor that is recommended and may be essential when working with low amounts of starting RNA.

Reverse transcription and thermal cycling:
Place the vials into a real-time PCR cycler and start the following program.
 

Protocol for standard PCR cycler combined with gel - based DNA analysis the following cycling protocol is recommended:

²⁾ The annealing temperature depends on the melting temperature of the primers.
³⁾ The elongation time depends on the length of the amplicon. A time of 1 min for a fragment of 1,000 bp is recommended.

Note:
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary. Note that optimal reaction times and temperatures should be adjusted for each particular sample/primer pair.