Navigation Links
Your Data: Detection of Oocyte-specific Gene Expression Using Ambion's Cells-to-cDNA Kit


Ambion's Cells-to-cDNA Kit produces cDNA ideal for RT-PCR based gene quantitation, including experiments performed using real-time RT-PCR. The Cells-to-cDNA Kit comes in an easy-to-use format that produces first-strand cDNA from cultured cells without RNA isolation in less than 2 hours. By using an RT-compatible Cell Lysis Buffer that inactivates RNases, RT-PCR can be carried out on only a few cells.

Using the Cells-to-cDNA Kit, Dr. Alex Bortvin from the Whitehead Institute (Cambridge, Massachusetts) has successfully carried out RT-PCR amplification of an oocyte-specific gene and a housekeeping gene from individual mouse oocytes. Dr. Bortvin made some modifications to the Cells-to-cDNA protocol for this application. One fertilized oocyte (in 1 l of culture medium) was added to 5 l of Cell Lysis Buffer and overlaid with oil to prevent evaporation. After cell lysis, DNase I treatment was carried out with a 1:10 dilution of the enzyme at 37C for 30 min. cDNA was synthesized according to the Cells-to-cDNA protocol.

In order to ensure that clean templates for cDNA library synthesis and multiplex PCR applications was obtained, Dr. Bortvin purified the first-strand cDNA using a PCR purification kit (note that this should not normally be necessary). This step enabled him to use the PCR components and conditions that he had previously optimized for his gene of interest. PCR was carried out with gene-specific primers for 50 cycles. A "minus-RT" control reaction was included to rule out the presence of any trace genomic DNA contamination; none was seen (data not shown).

As shown in Figure 1, (top, right) using the described protocol, Dr. Bortvin was able to easily detect both his gene-specific transcript and the GAPDH housekeeping mRNA from single oocytes using the Cells-to-cDNA Kit protocol.

Figure 1. Gene-specific RT-PCR on a Single Mouse Oocyte Using the Cells-to-cDNA Kit. 10 l of each PCR was electrophoresed for 25-30 minutes in a 2% agarose gel in 1X TBE buffer containing ethidium bromide.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. Your Data: Use of pSilencer 1.0-U6 siRNA Expression Vector in the Analysis of Synaptotagmin Location and Function
2. Your Data: RNAlater Stories
3. Your Data: Silencing Hepatitis C Virus Replication With In Vitro Transcribed siRNAs
4. Your Data: Silencing Neural Cell Adhesion Genes with In Vitro Transcribed siRNAs
5. Your Data: Silencing of a Pro-apoptotic Transcription Factor Using Synthetic siRNA
6. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
7. A New PCR-based Mycoplasma Detection Method
8. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
9. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
10. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
11. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/22/2017)... ... February 22, 2017 , ... ... development of precision treatments for neurodegenerative diseases, today announced it has issued a ... approach.” This is one of a series of commentaries from ProMIS’s scientific team ...
(Date:2/22/2017)... , Feb 22, 2017 Dublin ... the "Global Biological Crop Protection (Bio-Pesticide) Market-By Type, By Application, ... report to their offering. ... Global Biological Crop Protection Market is forecasted to ... The strong growth in biopesticide or biological crop protection market is ...
(Date:2/22/2017)... ... February 22, 2017 , ... NDA Partners ... MD former Acting Deputy Director in the FDA CDRH Division of Cardiovascular, Respiratory, ... joined the company as an Expert Consultant. , In Dr. Spyker’s accomplished career, ...
(Date:2/22/2017)... N.C., Feb. 22, 2017  United Therapeutics Corporation (NASDAQ: ... results for the fourth quarter and year ended ... results reflect continued growth as net revenues reached ... Martine Rothblatt, Ph.D., United Therapeutics, Chairman and Chief ... to develop and advance our growing product pipeline, ...
Breaking Biology Technology:
(Date:2/7/2017)... Minn. , Feb. 7, 2017   MedNet ... supports the entire spectrum of clinical research, is pleased ... iMedNet , its innovative, highly flexible and award ... iMedNet customers. iMedNet is a proven ... provides Electronic Data Capture (EDC), but also delivers an ...
(Date:2/3/2017)... , Feb. 3, 2017 A new independent ... Strategy Partners, LLP (IdSP) . Designed to fill a ... complex identity market, founding partners Mark Crego ... combined years just in identity expertise that span federal ... non-profit leadership. The Crego-Kephart combined expertise has a common ...
(Date:1/31/2017)... Jan. 31, 2017  Spero Therapeutics, LLC, a ... the treatment of bacterial infections, today announced it ... candidates from Pro Bono Bio Ltd (PBB) to ... multi-drug resistant forms of Gram-negative bacteria.   The assets ... Ltd, a PBB group company. "The ...
Breaking Biology News(10 mins):