RNAi transfection

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Cell biologist
Cell biologist's picture
RNAi transfection

Hi everyone,

I am trying to transfect RNAi in MCL cell line. Can anyone give me a efficient protocol for this except lypofectamin. I have used it and did not get a satisfactory result.
Thanks

Jason King
Jason King's picture
If MCL are mantle cell

If MCL are mantle cell lymphomas then the paper below might be interesting. It seems as though lentivirus vectors are commonly used to express shRNAs in MCL lines but for oligonucleotide (and by extension, siRNA) transfection, the authors used lipofectamine. Maybe it needs some more optimization than for cells that are relatively easy to transfect.

Blood. 2009 Jan 8;113(2):396-402. Epub 2008 Oct 21.
MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes.
Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada K.

Piece from methods:

Antisense oligonucleotides (ASOs) and their respective scrambled oligonucleotides (SCOs) were synthesized as hybrid deoxyribonucleotide molecules linked between the 2'-O and the 4'-C-methylene bridge (locked nucleic acid, LNA) modification of G and C residues (Greiner, Tokyo, Japan). The ASOs used were as follows: AS-miR-17 (AS-17), 5'-ACTACCTGCACTGTAAGCACTTTG-3'; AS-miR-20a (AS-20), 5'-CTACCTGCACTATAAGCACTTTA-3'; and AS-miR-19a (AS-19), 5'-TCAGTTTTGCATAGATTTGCACA-3'. The SCOs used were as follows: SC-miR-17 (SC-17), 5'-TAACGTCACTTCGACTGAACTGCT-3'; SC-miR-20a (SC-20), 5'-ATCTCATACTACACTTGAACACT-3'; and SC-miR-19a (SC-19), 5'-GTCTATTGGTATATCTAACYGCA-3'.

Cells were plated to a density of 105 cells/well in 24-well dishes on day 1, and then transfected with oligonucleotides (20 nM) using Lipofectamine 2000 (day 2). Two days later (day 4), the cells were harvested and analyzed. Alternatively, cells were plated to a density of 105 cells/well in 6-well plates on day 1, after which the cells in triplicate wells were transfected with oligonucleotide (20 nM) using Lipofectamine 2000 on day 2. For cell-cycle analysis, the cells were suspended in a mixture containing 0.2 mL 0.9% NaCl and 3 mL 70% EtOH, and the nuclei were stained with propidium iodide (Sigma-Aldrich). Cellular DNA content was measured using a FACSCalibur flow cytometer equipped with the CellQuest program (BD Biosciences, San Jose, CA).

Cell biologist
Cell biologist's picture
Thank you so much for the ref

Thank you so much for the ref.
Yes! MCL is mantle cell lymphomas.
Did you use this method?
I'll try this.