RNA / RT-PCR

VolcanoCell2G RT-PCR 2x Master Mix

(available with or without separate lysis buffer included)

This mix has been specifically optimized for direct-from-cell applications.

Skip the time-consuming and expensive RNA purification. The VolcanoCell2G RT-PCR 2x Master Mix enables you to quantify and detect RNA directly from a cell suspension, or a cell lysate, without a need of purification steps. Simply detach the cells, add the cell suspension directly to your RT-PCR reaction mix, place in your real-time instrument, and start.

Optionally, use our newly established lysis buffer to lyse the cells directly in the well or flask, simply spin down and add the supernatant directly to your RT-PCR mix. - Ready.

The lysates can also be frozen and stored at -20°C.

 

Article Product Size Price  
#7100S VolcanoCell2G RT-PCR 2x Master Mix 100 reactions á 25 µl, 1 x 1.25 ml € 180,- will be soon discontinued
#7100M VolcanoCell2G RT-PCR 2x Master Mix 500 reactions á 25 µl, 5 x 1.25 ml € 780,- will be soon discontinued
#7101S VolcanoCell2G RT-PCR 2x Master Mix (including separate lysis buffer) 100 reactions á 25 µl, 1 x 1.25 ml € 180,- new
#7101M VolcanoCell2G RT-PCR 2x Master Mix (including separate lysis buffer) 500 reactions á 25 µl, 5 x 1.25 ml € 780,- new

Bulk offer: Should you require larger amounts (> 10 ml), significant discounts are available. Please contact us to receive an individual quotation.

 

download-pdf Product Manual (PDF) #7101Order

download-pdf Product Manual (PDF) #7100

 

 

Applications

Applications

  • Direct from cell RNA detection and quantification using primers/probe or a realtime PCR dye
  • Gene expression analysis on 100-10.000 cells per sample.
  • Suitable for a wide-range of cell types

Example Results

VolcanoCell2G allows sensitive RNA detection, direct from cells: 

a)

b)

Figure 1. 0-step direct from cells RT-qPCR

a) Workflow of the 0-step direct from cells RT-qPCR is easier and faster than the 2-step standard RT-qPCR. b) GAPDH mRNA was detected from Lund human mesencephalic (LUHMES) cells, which were resuspended according to the VolcanoCell2G protocol (with separate lysis buffer included).

Reaction Setup

Reaction setup

Component Volume Final concentration
VolcanoCell2G RT-PCR 2x Master Mix 12.5 µl 1x
Primer forward (10 µM)* 1 µl 0.4 µM (0.05-1 µM)
Primer reverse (10 µM)* 1 µl 0.4 µM (0.05-1 µM)
Probe (10 µM) 1 µl 0.4 µM (0.05-1 µM)
Template**:
Cell suspension (in nuclease-free water)***
or
cell lysate (in 1x lysis buffer)***

1 - 9.5 µl

2.5 µl

<10.000 cells/reaction

<10.000 cells/reaction
Nuclease-free water up to 25 µl total reaction volume  

Keep all components on ice.
Spin down and mix all solutions carefully before use.

*Please note that Volcano RT-PCR mix is amplifying from DNA and RNA templates, so the use of RNA-selective primers, which are binding onto exon-exon junctions only, is mandatory.

**Suggested cell number is 10 - 10.000 cells suspended in nuclease-free water or 1x VolcanoCell2G lysis buffer. Cell suspensions should be prepared fresh as suggested in the METHODS section of the manual enclosed.

***Template from cell cultures can be prepared either directly by diluting cell pellet in nuclease-free water (Protocol 1 in the METHODS section of the product manual) or by lysing cells in the well or tube with 1x VolcanoCell2G lysis buffer (Protocol 2 in the METHODS section of the product manual) which may be more useful for high-throughput assays when RT-PCR reactions need to be prepared from separate wells (eg.: on a microtiter plate).

 

 

0-step RT-PCR

Typical 0-step RT-PCR protocol (an isothermal reverse transcription step is not needed)

Step Temperature Time
Initial denaturation 95°C 3 min
Denaturation 95°C 15 sec
Annealing/Extension* 55-75°C 60 sec   (25-40 cycles)
Hold <10°C hold

*Typically, the annealing temperature is about 3-5°C below the calculated melting temperature of the primers used. A new PCR is ideally established by running a temperature gradient in order to find the best annealing/extension temperature for each primer pair. Also a three-step protocol can be applied with separate annealing and extension steps.

 

Detailed product description

The kit contains an engineered, extremely thermostable reverse transcriptase and combined DNA polymerase, including a specially optimized buffer system that requires only a small amount of cells (100 to 10.000 cells). A separate lysis buffer is included in the kit as well.

The included Volcano2G DNA polymerase was engineered through multiple generations of directed, artificial evolution and has a half-life at 95°C of >40min. It facilitates a “zero-step” RT-PCR directly from cell suspension or lysate without an isothermal reverse transcription step.

VolcanoCell2G RT-PCR 2x Master Mix contains all components necessary for a successful and reliable RT-PCR in all standard real-time cycler instruments and is compatible with the common RT-PCR detection methods such as fluorescent realtime PCR dyes or fluorogenic probes.

Recommended amplicon size should be between 60-300 bp.


Please note that primers which span an exon-exon junction must be used in order to limit the amplification only to mRNA targets.

By avoiding expensive, time-consuming cell lysis or RNA purification, VolcanoCell kit is ideal for high-throughput applications, RNAi knockdown or gene expression experiments.The pre-ready 2x mix ensures repeatable RT-PCR results, significantly reduces set-up times and the risk of pipetting mistakes. Only primers and template need to be added. Cell lysates can be easily prepared with the included lysis buffer. However, amplicon size should ideally be between 60-300 bp!

 

Composition / Content of the product

VolcanoCell2G RT-PCR 2x Master Mix contains an engineered DNA polymerase with a reverse transcription activity in an application-optimized buffer system with dNTPs. A separate lysis buffer is also included.

 

Quality Control

RT-PCR activity: VolcanoCell2G RT-PCR 2x Master Mix was tested for a successful RT-PCR performance. A 151 bp fragment (HPRT1 mRNA) was amplified from human total RNA extract in a realtime PCR cycler and visualized as a single amplified product.

DNA polymerase activity: Volcano2G polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and DNA primer.
Enzyme-concentration has been determined by protein-specific staining. Please inquire more information at info@mypols.de for the lot-specific concentration.

 

Storage

This product is shipped on cool packs. Please store the product upon arrival at -20°C.
 Minimize the number of freeze-thaw cycles by storing in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C.

 

References

Chovancova, P., Merk, V., Marx, A., Leist, M., Kranaster, R. (2017) Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a ‘zero-step’ RT-qPCR protocol.Biol Methods Protoc (2017) 2 (1): bpx008. doi: https://doi.org/10.1093/biomethods/bpx008

 

Blatter, N., Bergen, K., Nolte, O., Welte, W., Diederichs, K., Mayer, J., Wieland, M. and Marx, A. (2013), Structure and Function of an RNA-Reading Thermostable DNA Polymerase.Angew. Chem. Int. Ed., 52: 11935–11939. doi:10.1002/anie.20130665.

 

Kranaster, R., Drum, M., Engel, N., Weidmann, M., Hufert, F. T. and Marx, A. (2010), One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase.Biotechnology Journal, 5: 224–231. doi:10.1002/biot.200900200.

VolcanoCell_teaser

 

img-faqsFAQ`s

How can I design RNA-specific primers?

 You can use one of the free primer design tools in the internet, such as primer-blast on the homepage: www.ncbi.nlm.nih.gov/tools/primer-blast/. Ensure, that you select the option: "primers must span an exon-exon junction". This is useful for limiting the amplification only to mRNA, as Volcano poylmerase will amplify from any nucleic acid target consisting of both RNA or DNA. However, you can use standard primers if you want to amplify mRNA as well as the corresponding genomic DNA.

I am only getting primer dimers. How can I establish my RT-PCR?

- Keep all reagents during PCR setup on ice.
- Programm the PCR protocol on your cycler first, before you setup the PCR mix and if applicable preheat the lid of your cycler.
- When you do a new PCR for the first time, start with our standard protocol and recommended reaction setup concentrations.
- Run a temperature gradient to identify the best temperature yielding in the cleanest product. This temperature is only limited by the primer annealing temperature, as Volcano polymerase is thermostable.- Use a "multiple primer analyzer" to test your primer sequences for self-complementarity, secondary structures and primer cross annealing. 

Is the VolcanoCell2G RT-PCR 2x Master Mix applicable for procaryotes and yeasts?

An application for procaryotes and yeasts is not yet established for the VolcanoCell2G RT-PCR 2x Master Mix. As procaryotes do not have exon-exon spans, a DNase I treatment of your supernatant is necessasy for amplification of mRNA only.


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