Introduction:
The study of gene expression often needs RNA preparation followed by cDNA synthesis and PCR. However,
difficulty in obtaining a large number of cells for RNA preparation is the bottleneck in some of the studies,
such as laser dissected samples, FACS sorted cells, the cultured cells in 96-wells, and liquid biopsy. Cell
numbers are limited (<2,000 cells), which restrict RNA preparation. AffiGEN’s Direct cDNA cell lysis buffer
allows preparation of cell lysate, which can be used for direct reverse transcription without RNA preparation.
Cell lysate made from a few cells is good enough for reverse transcription of RNA to cDNA and following
traditional PCR and real time PCR analysis or cDNA plate array assays.
Materials provided:
10 mL of Cell Lysis Buffer for direct cDNA Synthesis
Materials that may be required but are not provided:
AffiCHEM® 1st Strand cDNA Synthesis Kit (Cat# AFG-KLE-009134)
AffiPCR® One Step SYBR Green RT-PCR Mix (Cat# AFG-EKP-001)
AffiZYME® Deoxyribonuclease I (Cat# AFG-KLE-020230)
Sample preparation procedure:
1. For cell numbers 1–500: Add five times the volume of 1X Cell Lysis Buffer to the cell suspension (e.g.,
mix 30 μL of cell suspension with 150 μL of 1X Cell Lysis Buffer). For 500–2000 cells: Spin down the cells,
discard the supernatant, leaving 30–40 μL of the pellet. Then, add 150–200 μL of 1X Cell Lysis Buffer to
lyse the cells. If fewer than 2000 cells are used, skip the PBS wash step. For 2000–10,000 cells: Wash the
cells with 200 μL of PBS, remove the PBS, and add lysis buffer at five times the volume of the pellet. For
all cell numbers: Perform 2–4 freeze-thaw cycles to aid in cell lysis and minimize inhibitors. * Note: Keep
the cells on ice during the procedure to prevent cells from degrading. **
2. Incubate in Cell Lysis buffer for 10 minutes. Remove contaminated DNA by spinning the sample at
12,000rpm for 5 minutes. Optional: Add 0.25-1 μL DNase I and incubate at 37 oC for 10 minutes and
inactivate at 75 oC for 10 minutes.
3. Transfer the supernatant to a fresh tube. Heat at 75 oC for 10 minutes and put on ice. The cell lysate is
ready for use or can be stored at -80 oC for the future usage.