_{One.Direct.Step RT-qPCR Kit for Probes (without ROX; with high-ROX)}

_{Direct RT-PCR: Amplification directly from blood or cell material -} 

Safe time, increase your result

_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

• Simultanly detection of multiple targets (multiplex PCR)

Description:

The kit is recommended for use with Dual Labelled 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 impurified 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! Easy protocol.

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

The Kit is used for reverse transcription and recommended for fluorescent probes with TaqMan, 

Molecular Beacons etc.

Kit content:

1.Extraction Buffer: 1x concentrated

2.Direct Enzyme: Mix of engineered reverse transcriptase, mAB-inhibited hot start polymerase, dNTPs, reaction buffers, enhancers and additives

PCR-grade Water

3.Optional: ROX Dye

Easy Preparation (e.g. 50 µl reaction volume)

1. Whole Blood (not treated heparin-, EDTA- or citrate-treated whole blood)

• Add 2-5 µl whole blood (for 50 µl reaction volume) without any pre-treatment directly to the RT-PCR assay.

2. Swab Samples from nasal or swaps

• fill 200 µl Extraction Buffer in a 1,5 ml Tube

• Cut the tip with nasal or throat swap and put to micro tube and vortex about 15-20 sec

• let absorb and incubate at room temperature for about 3 min

• press the tip of the swap to the wall of the 

_{m​icrotube  and take it out}

• centrifuge the tube extensively and transfer, for a 50µl reaction volume, 2-5 µl of the supernatant to your RT-PCR assay.

3. Animal or Plant Tissue samples

• Prepare a small piece from animal or plant tissue not

exceeding 8 mm in diameter.

• Crack plant seeds to less than 1 mm in diameter using a cell-disrupter, Tissue-lyser or small hammer.

• Add 1x Extraction Buffer to the tissue sample using:

 Sample size

1-2 mm - 50 µl

3-4 mm - 100 µl

5-8 mm - 200 µl

• mix Extraction Buffer and sample briefly and

 incubate for about 3 min at room temperature

Centrifuge extensively and and transfer 2-5 µl (for

 _{50 µl reaction volume) of the supernatant to your} RT-qPCR assay

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.}

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

_{RT-qPCR enzyme mix 2x   1x             10 µl              25 µl}

Sample, whole

_{blood or extracted - - 1-2 µl             2-5 µl}

_{Forward primerXn 1.) 10 µM 300 nM 0,6 µl 1,5 µl}

_{Reverse PrimerXn 1.)          10 µM 300 nM 0,6 µl              1,5 µl}

_{Dual labelled probe / TaqMan Xn 1.)               10 µl 200 nM 0,4 µl              1 µl}

_{optional ROX                                         25 µM   500nM            0,4 µl      1 µl}

_{PCR- grade water                                    -            -            up to 20 µl up to 50 µl}

1.) for each PCR-Target (Multiplex PCR) take Xn+1 primers and the related amount of TaqMan probe

 Note: For each primer on have to optimize the best assay parameters. The optimal primer can vary from 100 - 500 nM.

Reverse transcription and thermal cycling:

Place the vials into a real-time PCR cycler and start the following program

reverse transcription                50-55 °C 10-15 min      1x

initial denaturation                95 °C         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 2.)           50 °C             up to 30 min       1x}

 _{initial denaturation                95 °C             3-5 min       1x}

_{denaturation                         95 °C  15 sec       35-45x}

_{annealing 3.) 4.)              55-65 °C )            1 min 3)     35-45x}

_elongation                       67 °C           1 min/kb       35-45x

_{final elongation              67 °C             5 min        1x}

2.) 10 min for  amplicons < 200 bp; each 100 bp fragment length need about 3 min longer incubation time

3.)The annealing temperature depends on the melting temperature of the primers.

4.) 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.}