NDLI: Improved quantitative real-time RT–PCR for expression profiling of individual cells

Content Provider	PubMed Central
Author	Liss, Birgit
Copyright Year	2002
Abstract	The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT–PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies.
Starting Page	89
File Format	PDF
ISSN	13624962
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	17
Volume Number	30
Language	English
Publisher	Oxford University Press
Publisher Date	2002-09-01
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Improved quantitative real‐time RT–PCR for expression profiling of individual cells

Development and Validation of Endogenous Reference Genes for Expression Profiling of Medaka (Oryzias latipes) Exposed to Endocrine Disrupting Chemicals by Quantitative Real-Time RT-PCR

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR

Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR

Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

Improved quantitative real-time RT–PCR for expression profiling of individual cells

Similar Documents

Improved quantitative real‐time RT–PCR for expression profiling of individual cells

Development and Validation of Endogenous Reference Genes for Expression Profiling of Medaka (Oryzias latipes) Exposed to Endocrine Disrupting Chemicals by Quantitative Real-Time RT-PCR

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR

Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR

Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

Improved quantitative real-time RT–PCR for expression profiling of individual cells