Use of RNA amplification in the optimal characterization of global gene expression using cDNA microarrays

Abstract

Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.

Characterization of a method for profiling gene expression in cells recovered from intact human prostate tissue using RNA linear amplification

“…Although this approach has previously been described, much higher amounts of starting RNA were used. 10,42,43 It was therefore important that we went on to validate and characterize the current methodology, demonstrating its ability to measure accurately differences in gene expression between different cell types starting with low nanogram amounts of RNA. As this is the amount of RNA typically recovered from a single-cell population extracted from intact tissue, it was also important that we then went on to demonstrate its practical applicability in this regard.…”

“…[23][24][25]42,47,48 As the efficiency of transcription is not equal for all genes, one would expect to find a low correlation. It is possible that apparent differences reported in the literature relate to some extent to including genes in the analysis, which are only expressed at trivial levels.…”

Ativação de genes apoptóticos no bloqueio do desenvolvimento em embriões bovinos

“…Apesar destas constatações, os resultados obtidos indicam que o uso de RNA antisenso amplificado mostrou ser um protocolo adequado à hibridização quando a amostra biológica for escassa, obtendo resultados satisfatórios em micro arranjos coonfirmados por análise da expressão gênica por PCR quantitativo em Tempo Real (ELLESTAD et al, 2006;JENSON et al, 2003;LI et al, 2004;MAMO et al, 2006a;PARK et al, 2004;STOYANOVA et al, 2004;ZHU;BABA, 2006).…”

“…(BAUGH et al, 2001;GOMES et al, 2003;JENSON et al, 2003;LI et al, 2004;PARK et al, 2004;PATEL et al, 2005;STOYANOVA et al, 2004;WANG E. et al, 2000;WANG, 2005;ZHU;BABA, 2006 (LI et al, 2004;ROBERT et al, 2002;PATEL et al, 2005). As hibridações foram realizadas com estas mesmas amostras de RNA, porém, com uma terceira etapa de amplificação, confirmando uma maior modulação destes genes em embriões L8, similar ao demonstrado com apenas uma única fase de amplificação.…”

“…Devido à ocorrência de apoptose espontânea apenas após a AGE em embriões bovinos, este bloqueio do desenvolvimento parece ser um mecanismo ativo, baseado no balanceamento entre proteínas pró e anti-apoptóticas, mas não envolvendo os membros da família Bcl-2 (BADR et al, 2007;FABIAN et al, 2007;GJORRET et al, 2003;JURISICOVA et al, 1998;MATWEE;KING, 2000;MEIRELLES et al, 2004;OPIELA et al, 2008;YANG & RAJAMAHENDRAN, 2002). FAIR et al, 2007;GINSBERG, 2005;GOMES et al, 2003;JENSON et al, 2003;JEONG et al, 2006;JONES et al, 2008;KATO et al, 2007;MAMO et al, 2006aMAMO et al, , 2006bMISIRLIOGLU et al, 2006;PATEL et al, 2005;SCHNEIDER et al, 2004;SIRARD et al, 2005;STOYANOVA et al, 2004;ZHU;BABA, 2006 Gelder e colaboradores (1990) e posteriormente repetida por outros laboratórios (ELLESTAD et al, 2006;GINSBERG, 2005;JENSON et al, 2003;JEONG et al, 2006;MAMO et al, 2006a;WADENBÄCK et al, 2005;WANG E. et al,2000).…”