HotScriptase RT Cell Mastermix >:
For the cDNA synthesis directly from cells, there is no need for a time-consuming and expensive cell lysis or RNA purification any more.
Our new HotScriptase RT Cell Mastermix > llows simultaneous reverse transcription and amplification of cells or cell suspensions quickly and reliably without prior isolation of mRNA. Simply add your cells directly into the PCR reaction and start.
Only approx. 100 - 10,000 cells per batch are required. Additionally, there is no isothermal step necessary by the use of the highly thermostable HotScriptase RT Polymerase > in the mastermix. The complete essay can be started immediately very hot. The RT reaction takes place during the "normal" extension. This proves to be beneficial just in cDNA synthesis.
The entire process is thus greatly shortened, thereby a premature disintegration of the sensitive and valuable RNA is counteracted.
RT-PCR with simultaneous realtime PCR:
The Quantification and Detection of RNA is possible directly from the cell suspension without separate lysis or additional purification steps. The mastermix is especially suitable for short fragments between 60 and 300bp.
In PCR directly from cells, especially from blood, the cellular material normally disturbs the realtime PCR. With the HotScriptase RT Cell Mastermix this is no problem any more: simply increase the amount of fluorescent dye (eg, use SYBR® Green up to 1:30 instead of 1:1), then a signal can be detected again.
We recommend the use of RNA-specific primers that bind in the exon-exon region to exclude the amplification of genomic DNA contained in the cell mixture. Genomic DNA is usually included in the cell suspension. If unspecific primers are used, you obtain a standard PCR instead of a RT-PCR.
The Genaxxon HotScriptase RT Cell Mastermix > is ideally suited for high-throughput applications, RNAi knockdown, realtime PCR or gene expression and in "Next Generation Sequencing".
Protocol for direct RT-PCR from cells >
For adherent cell cultures: follow all steps below.
For suspension cultures: skip steps 1-4 and start with step 5.
1. Remove media from cell culture dish/flask and add an appropriate volume of 1x PBS (use 10mL PBS for 10cm dish or T75 flask) to wash the cells.
2. Remove the PBS and add trypsin (or better Accutase – cat#: C4356) to detach your cells (use enough trypsin to cover the cells e.g 1mL trypsin for a 10cm dish or 3mL trypsin for a T75 flask).
3. Incubate at 37°C or at room temperature and check cell detachment under a microscope every 2 min until you see the cells have all detached.
4. Once the cells have detached, add medium containing serum to inactivate trypsin (for each 1mL of trypsin add 2mL of serum containing medium).
5. Transfer the cell suspension to a centrifuge tube and centrifuge at 1300rpm for 3 min at room temperature to pellet the cells.
6. Remove the supernatant and resuspend the cell pellet in 1mL-10mL 1xPBS according to the size of the pellet to determine the total cell count (this will also wash the cells and get rid of any remaining medium).
7. Count the cells from ~10µL of the cell suspension using a hemocytometer counting chamber or an electronic counter.
8. Centrifuge again at 1300rpm for 3 min at room temperature to pellet the cells.
9. Now prepare the template for your PCR reaction: Aspirate the supernatant and resuspend the pellet in an appropriate amount of nuclease-free water to achieve cell density of max. 5000 cells/μL (for a final cell number of 10.000 cells/reaction). Alternatively, prepare several tubes of different cell densities ranging from 50 to 5000 cells/μL (100-10.000 cells/reaction) in order to investigate the optimal number of cells per reaction for your future experiments. Keep the suspensions on ice and always prepare fresh before PCR.