Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA

Analytical Biochemistry
Mads BakNiels Tommerup

Abstract

Several methods have been developed for amplification of RNA, making it possible to use cDNA microarrays for analysis of samples limited in amount of total RNA. The most widely used amplification protocol, the Eberwine method, amplifies RNA in a linear manner through in vitro transcription (IVT). However, when starting material is limited to nanogram amounts of total RNA, several rounds of amplification are necessary, making this method both expensive and labor-intensive. Amplification by PCR is robust and is able to amplify extremely limiting material. However, it is possible that the nonlinear nature of PCR could result in reduced reproducibility of the amplification compared with IVT. We have evaluated two methods that use a combination of PCR and IVT for amplification of nanogram amounts of total RNA. We have compared microarray results obtained by these methods with results obtained by two established methods: indirect labeling of 20 microg total RNA and Eberwine amplification of 1 microg total RNA. Starting from as little as 5 ng of total RNA, both methods yielded results in concordance with the Eberwine method.

References

Mar 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·R N Van GelderJ H Eberwine
Feb 26, 1999·Nucleic Acids Research·M MatzA Chenchik
Feb 27, 2001·Nucleic Acids Research·L R BaughC P Hunter
Jun 29, 2002·Nature Biotechnology·Charlie C XiangMichael J Brownstein
Feb 1, 2003·Biochemical and Biophysical Research Communications·Kazuhiko AoyagiHiroki Sasaki
Apr 25, 2003·Nucleic Acids Research·Charlie C XiangMichael J Brownstein
Nov 5, 2003·Methods : a Companion to Methods in Enzymology·Gordon K Smyth, Terry Speed
Apr 13, 2004·Proceedings of the National Academy of Sciences of the United States of America·Andrew I SuJohn B Hogenesch
Nov 24, 2004·Biochemical and Biophysical Research Communications·Jik Young ParkSuk Woo Nam
Dec 23, 2004·American Journal of Physiology. Cell Physiology·Ruchira SinghPhiline Wangemann
Mar 3, 2005·Genome Research·Chang Gue SonJaved Khan

Citations

Jul 22, 2009·BMC Genomics·Julie E LangChristopher Haqq
Nov 15, 2011·The Journal of Molecular Diagnostics : JMD·John S KaddisTimothy J Triche
Jul 19, 2012·PloS One·Nancy A StearnsDavid S Pisetsky

Related Concepts

Liver
Lung
RNA
Transcription, Genetic
Nested Polymerase Chain Reaction
cRNA Probes
Cdna Microarrays
Nucleic Acid Amplification Tests

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Alzheimer's Disease: MS4A

Variants within the membrane-spanning 4-domains subfamily A (MS4A) gene cluster have recently been implicated in Alzheimer's disease in genome-wide association studies. Here is the latest research on Alzheimer's disease and MS4A.

Pediculosis pubis

Pediculosis pubis is a disease caused by a parasitic insect known as Pthirus pubis, which infests human pubic hair, as well as other areas with hair including eye lashes. Here is the latest research.

Rh Isoimmunization

Rh isoimmunization is a potentially preventable condition that occasionally is associated with significant perinatal morbidity or mortality. Discover the latest research on Rh Isoimmunization here.

Genetic Screens in iPSC-derived Brain Cells

Genetic screening is a critical tool that can be employed to define and understand gene function and interaction. This feed focuses on genetic screens conducted using induced pluripotent stem cell (iPSC)-derived brain cells. It also follows CRISPR-Cas9 approaches to generating genetic mutants as a means of understanding the effect of genetics on phenotype.

Enzyme Evolution

This feed focuses on molecular models of enzyme evolution and new approaches (such as adaptive laboratory evolution) to metabolic engineering of microorganisms. Here is the latest research.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Pharmacology of Proteinopathies

This feed focuses on the pharmacology of proteinopathies - diseases in which proteins abnormally aggregate (i.e. Alzheimer’s, Parkinson’s, etc.). Discover the latest research in this field with this feed.

Alignment-free Sequence Analysis Tools

Alignment-free sequence analyses have been applied to problems ranging from whole-genome phylogeny to the classification of protein families, identification of horizontally transferred genes, and detection of recombined sequences. Here is the latest research.