Description(s):

The study of gene expression often needs RNA preparation followed by cDNA synthesis and PCR, but most of the time, you don't want to waste a large amount of cells for RNA preparation. This is a particular problem for researchers using laser dissected samples, FACS sorted cells, cultured cells in 96-wells, and liquid biopsy. Signosis’ Direct cDNA cell lysis buffer allows researchers to prepare cell lysate from small samples, which can be used for direct reverse transcription without RNA preparation. Cell lysate made from even a few cells is good enough for reverse transcription of RNA to cDNA, which you can then use for PCR analysis or cDNA plate array assays.

Benefits:

Simplified procedures - Reverse transcription can be performed in cell lysate without RNA preparation. Limited cell numbers required - A few cells are good enough to make cDNA for PCR.

Principle

The buffers are optimized for cell lysate preparation and real-time RT-PCR.

 

Data

Signosis Direct cDNA cell lysis buffer. A: The indicated cells were lysed with Direct cDNA cell lysis buffer from Signosis and competitor respectively, and subjected to RT-PCR for ß-actin with 30 cycles. B: Testing for genomic DNA contamination. Lane1. ß-actin was amplified with 36 PCR cycles directly from cell lysate without reverse transcription (RT). Lane2. ß-actin was amplified with 36 PCR cycles directly from cDNA transcribed from cell lysate.     Comparison of PCR products amplified from lysates made with Signosis Direct cDNA cell lysis buffer or TRIzol without DNAse treatment.  By using intron-spanning primers, DNA amplified from genomic DNA and reverse-transcribed mRNA can be distinguished.  PCR amplification from RNA prepared with TRIzol contains a band that corresponds with the predicted length of the genomic DNA, as well as the predicted length of the mRNA. However, PCR amplification from cell  lysate prepared with Signosis Direct cDNA cell lysis buffer only contains a band that corresponds with the predicted length of the mRNA and not the genomic DNA contamination.

Literature

View user manual

Citations

 

1.Inositol 1,4,5-trisphosphate (IP3) receptor up-regulation in hypertension is associated with sensitization of Ca2+ release and vascular smooth muscle contractility. Abou-Saleh H, Pathan AR, Daalis A, Hubrack S, Abou-Jassoum H, Al-Naeimi H, Rusch NJ, Machaca K J Biol Chem. 2013 41958;288(46):32941-51. 

2.A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons. Zhao X, Tang Z, Zhang H, Atianjoh FE, Zhao JY, Liang L, Wang W, Guan X, Kao SC, Tiwari V, Gao YJ, Hoffman PN, Cui H, Li M, Dong X, Tao YX. Nat Neurosci. 2013 Aug;16(8):1024-31. 

3.Protein Kinase CK2 Inhibition Down Modulates the NF-κB and STAT3 Survival Pathways, Enhances the Cellular Proteotoxic Stress and Synergistically Boosts the Cytotoxic Effect of Bortezomib on Multiple Myeloma and Mantle Cell Lymphoma Cells. Manni S, Brancalion A, Mandato E, Tubi LQ, Colpo A, Pizzi M, Cappellesso R, Zaffino F, Di Maggio SA, Cabrelle A, Marino F, Zambello R, Trentin L, Adami F, Gurrieri C, Semenzato G, Piazza F. PLoS One.  2013 41544;8(9):e75280. 

4.IP3 receptor up-regulation in hypertension is associated with sensitization of Ca2+ release and vascular smooth muscle contractility. Abou-Saleh H, Pathan AR, Daalis A, Hubrack S, Abou-Jassoum H, Al-Naeimi H, Rusch NJ, Machaca K. J Biol Chem.  2013 Oct 4. [Epub ahead of print] 

5.Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows. Ho YK, Xu WT, Too HP. PLoS One.  2013 41522;8(9):e72463. 

6.Osteoprotegerin Contributes to the Metastatic Potential of Cells with a Dysfunctional TSC2 Tumor-Suppressor Gene. Steagall WK, Pacheco-Rodriguez G, Glasgow CG, Ikeda Y, Lin JP, Zheng G, Moss J. Am J Pathol.  2013 Sep;183(3):938-50. 

7.Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan® Low Density Array. Demarest TG, Murugesan N, Shrestha B, Pachter JS. J Neurosci Methods., 2012 ;206(2):200-4. 

8.Nuclear Aggregation of Olfactory Receptor Genes Governs Their Monogenic Expression.Clowney EJ, Legros MA, Mosley CP, Clowney FG, Markenskoff-Papadimitriou EC, Myllys M, Barnea G, Larabell CA, Lomvardas S. Cell., 2012 41222;151(4):724-37. 

9.Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus. Murugesan N, Paul D, Lemire Y, Shrestha B, Ge S, Pachter JS. Fluids Barriers CNS., 2012 41125;9(1):15. 

10. Alterations in Tight Junction Protein and IgG Permeability Accompany Leukocyte Extravasation Across the Choroid Plexus During Neuroinflammation.Shrestha, Bandana BS; Paul, Debayon MS; Pachter, Joel S. PhD . Journal of neuropathology and experimental …, 2014

11. Chronological in Vivo Imaging Reveals Endothelial Inflammation Prior to Neutrophils Accumulation and Lipid Deposition in HCD-Fed Zebrafish. Atherosclerosis. Luo, Hui, et al.  2019, doi:10.1016/j.atherosclerosis.2019.09.017.

12. N6‐Methyladenosine Demethylase FTO Contributes to Neuropathic Pain by Stabilizing G9a Expression in Primary Sensory Neurons. Li, Y., Guo, X., Sun, L., Xiao, J., Su, S., Du, S., Li, Z., Wu, S., Liu, W., Mo, K., Xia, S., Chang, Y.‐J., Denis, D. and Tao, Y.‐X. (2020). Adv. Sci.. doi:10.1002/advs.201902402

13. The transcription factor TAL1 and miR-17-92 create a regulatory loop in hematopoiesis. Meyer, A., Herkt, S., Kunze-Schumacher, H. et al. Sci Rep 10, 21438 (2020). https://doi.org/10.1038/s41598-020-78629-z

14. Downregulation of a Dorsal Root Ganglion-Specifically Enriched Long Noncoding RNA is Required for Neuropathic Pain by Negatively Regulating RALY-Triggered Ehmt2 Expression. Pan, Z., Du, S., Wang, K., Guo, X., Mao, Q., Feng, X., Huang, L., Wu, S., Hou, B., Chang, Y.‐J., Liu, T., Chen, T., Li, H., Bachmann, T., Bekker, A., Hu, H., Tao, Y.‐X. Adv. Sci. 2021, 2004515. https://doi.org/10.1002/advs.202004515