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:
Sample size (diameter) |
1-2 mm | 3-4 mm | 5-6 mm |
PCR-grade Water |
45 µl | 90 µl | 135 µl |
Extraction Buffer |
5 µl | 10 µl | 15 µl |
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.
component | stock conc. | final conc. | 20 µl assay | 50 µl assay |
direct reaction mix | 2x | 1x | 10 µl | 25 µl |
sample | - | - | 1-2 µl | 1-5 µl |
forward primer | 10 µM | 400 nM | 0,8 µl | 2 µl |
reverse Primer | 10 µM | 400 nM | 0,8 µl | 2 µl |
dual labeled probe | 10 µl | 200 nM | 0,4 µl | 1 µl |
direct enzyme mix 1) | 25x | 1x | 0,8 µl | 2 µl |
PCR- grade water | - | - | up to 20 µl | up to 50 µl |
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.
reverse transcription | 50 °C | 30 min | 1x |
initial denaturation | 95 °C | 3-5 min | 1x |
denaturation | 95 °C | 15 sec | 35-45x |
annealing and elongation | 60-65 °C 2) | 1 min 3) | 35-45x |
Protocol for standard PCR cycler combined with gel - based DNA analysis the following cycling protocol is recommended:
reverse transcription | 50 °C | 30 min | 1x |
initial denaturation | 95 °C | 3-5 min | 1x |
denaturation | 95 °C | 15 sec | 35-45x |
annealing | 55-65 °C 2) | 1 min 3) | 35-45x |
elongation | 72 °C | 1 min/kb | 35-45x |
final elongation | 72 °C | 5 min | 1x |
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.
- Direct RT-PCR: Amplification directly from blood or cell material - Safe time, increase your result.
- The RT-realtime PCR Kit is based on a genetically engineered reverse transcriptase;
- for improved efficiency, thermostability and specivicity; structured and long cDNA fragments; contains EvaGreen as intercalating dye and ROX as reference
Final price excl. shipping costs3