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:6/23/2016)... , June 23, 2016 A person commits ... the crime scene to track the criminal down. ... U.S. Food and Drug Administration (FDA) uses DNA evidence to ... Sound far-fetched? It,s not. The FDA has ... to support investigations of foodborne illnesses. Put as simply as ...
(Date:6/23/2016)... FRANCISCO , June 23, 2016   EpiBiome ... has secured $1 million in debt financing from Silicon ... ramp up automation and to advance its drug development ... its new facility. "SVB has been an ... beyond the services a traditional bank would provide," said ...
(Date:6/23/2016)... Calif. , June 23, 2016  Blueprint Bio, ... biological discoveries to the medical community, has closed its ... Matthew Nunez . "We have received ... with the capital we need to meet our current ... essentially provide us the runway to complete validation on ...
(Date:6/23/2016)... ... June 23, 2016 , ... ... line of intelligent tools designed, tuned and optimized exclusively for Okuma CNC machining ... Chicago. The result of a collaboration among several companies with expertise in toolholding, ...
Breaking Biology Technology:
(Date:6/21/2016)... VANCOUVER, British Columbia , June 21, 2016 ... been appointed to the new role of principal ... has been named the director of customer development. ... , NuData,s chief technical officer. The moves reflect ... development teams in response to high customer demand ...
(Date:6/16/2016)... 2016 The global ... reach USD 1.83 billion by 2024, according to ... Technological proliferation and increasing demand in commercial buildings, ... drive the market growth.      (Logo: ... development of advanced multimodal techniques for biometric authentication ...
(Date:6/9/2016)... control systems is proud to announce the introduction of fingerprint attendance control software, allowing ... are actually signing in, and to even control the opening of doors. ... ... ... Photo - http://photos.prnewswire.com/prnh/20160609/377487 ...
Breaking Biology News(10 mins):