Introduction
The EZ-press Cell to cDNA Kit offers a fast and easy method to produce cDNA from cells in culture without RNA isolation. cDNAs generated by this kit are suitable for gene cloning and quantitative analysis of gene expression, so it is a good substitution for the TRIzol method.
This kit offers several significant advantages compared with TRIzol method:
1. Easy to use. Starting from cells in culture, high quality cDNA can be synthesized in just two steps (cell lysis and reverse transcription), no RNA isolation is needed.
2. Fast. The whole experiment (from cells to cDNA) can be completed in less than 35 minutes.
3. Stable and highly reproducible. The total RNA is perfectly reserved throughout the experiment, enabling stable and highly reproducible results.
4. High sensitivity. High sensitivity. The lower detection limit for each sample is only 100 cells.
5. No Genome DNA left over. Genome DNA is removed adequately. Moreover, the reagents used in this kit are safe and non-toxic, which is pleasant compared with the smelly and hazardous TRIzol reagent.
Experimental Procedure at a Glance
Cell Lysis
Thaw the Cell Lysis buffer, invert or flick the tubes several times to mix thoroughly (do not use vortex), and place the tubes on ice.
1A. For adherent cells with a cell number of less than 3×105/sample:
a) Aspirate the culture medium from the wells.
b) Wash the wells with PBS (200 μl/well). Remove PBS as completely as possible without disturbing the cells.
c) Add Cell Lysis Buffer to each sample with the proportion of 80 μl Cell Lysis Buffer/1×105 cells（e.g. you should add 160ul Cell Lysis Buffer to 2×105 cells）, gently pipette up and down for 5 times to lyse the cells.
d) Incubate the cell lysates for 5 minutes at room temperature (19~27 °C).
1B. For adherent cells with a cell number of more than 3×105/sample or suspension cells:
a) (For adherent cells only, for suspension cells, start at step b) Detach cells using the subculturing method routinely employed in your laboratory.
b) Count then gently pellet the cells, discard the growth medium, and place cells on ice.
c) Wash cells in PBS by resuspending them in ~0.1 mL PBS per 1×105 cells.
d) Transfer a certain amount of cells(~1×105) to a new 1.5ml Eppendorf tube for each sample, centrifuge cells in low speed, then aspirate the PBS carefully without disturbing the cell pellets. Place the cells on ice.
e) Add 80 μl Cell Lysis Buffer to each sample, gently pipette up and down for 5 times to lyse the cells.
f) Incubate the cell lysates for 5 minutes at room temperature (19~27 °C).
2. Put the lysates on ice, carry out the RT reactions in 30 min.
Note: 1. The capacity of 80 μl Cell Lysis Buffer is about 1×105 cells, varies according to the cell type. To achieve the best results, it is strongly recommended to do a pilot experiment to determine the best number of cells per lysis.
2. Cells cultured in 96, 48, 24 and 12 well cell culture plates with the cell number of not more than 3×105 can be used for lysis directly after PBS washing. When doing with cells more than 3×105/well (or dish), follow the procedure 1B: cells must be detached first (step a). And then, add cell culture medium to the trypsin detached cells, count, low speed centrifuge and discard the supernatants (step b), resuspend with proper volume of PBS (step c), and then take ~1×105 cells/sample for lysis with 80 μl Cell Lysis Buffer (step d).
3. The cell lysate is incompatible with any other RT reagents commonly used (no or few cDNA can be produced using other RT reagents). So the cell lysates must be reverse transcribed using the specially optimized RT reagents supplied in this kit.
Reverse Transcription
Carry out Reverse Transcription as shown in the Product Manual.
Representative experimental results
cDNA synthesized by EZBioscience® EZ-press Cell to cDNA Kit and TRIzol - reverse transcription method were evaluated by real-time qPCR with 6 pairs of primers (in HEK-293T cells). Highly consistent results between EZ-press Cell to cDNA Kit and TRIzol -RT method can be observed in the following figure, which indicate that EZ-press Cell to cDNA Kit can completely replace TRIzol-RT method in preparing cDNAs from cells.
