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:2/11/2016)... 2016  Wellcentive today announced it has been ... -based community care organization (CCO) with more ... quality reporting and care management solutions and services. ... of quality managers, analysts and care managers while ... serving FamilyCare members. Oregon ...
(Date:2/11/2016)... Feb. 11, 2016   BioInformant announces the ... Cell Research Products, Opportunities, Tools, and Technologies – Market ... The first ... stem cell industry, BioInformant has more than a decade ... cell market, by stem cell type. This powerful 175 ...
(Date:2/10/2016)... MONTREAL , Febr. 10, 2016 /PRNewswire/ - BioAmber Inc. ... is pleased to announce that Mitsui & Co. Ltd., ... bio-based succinic acid plant, is investing an additional CDN$25 ... equity, increasing its stake from 30% to 40%.  Mitsui ... of bio-succinic acid produced in Sarnia ...
(Date:2/10/2016)... , Feb.10, 2016 ASAE is introducing a ... Management Companies (AMC) the option of joining or renewing ... fee determined by staff size, every employee in any ... ASAE and reap all available member benefits.   ... new organizational membership options will allow organizations of any ...
Breaking Biology Technology:
(Date:2/10/2016)... , February 10, 2016 ... According to 2016 iris recognition market ... iris recognition is more widely accepted for ... with both fingerprint and iris recognition technology ... user to avoid purchasing two individual biometrics ...
(Date:2/5/2016)... , Feb. 5, 2016 ... of the "Global Facial Recognition Market ... --> http://www.researchandmarkets.com/research/5kvw8m/global_facial ) has announced the ... Market 2016-2020" report to their offering. ... http://www.researchandmarkets.com/research/5kvw8m/global_facial ) has announced the addition of ...
(Date:2/3/2016)... -- Vigilant Solutions announces today that the ... solved two recent hit-and-run cases with the ... Solutions. Brian Wenberg explains, "I was ... out of a convenience store and witnessed an elderly male back out ... his vehicle and leaving the scene.  In his statement ...
Breaking Biology News(10 mins):