Ensuring good quality RNA for quantitative real-time PCR isolated from renal proximal tubular cells using laser capture microdissection.
BACKGROUND
In order to provide gene expression profiles of different cell types, the primary step is to isolate the specific cells of interest via laser capture microdissection (LCM), followed by extraction of good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous gene expression studies on specific cell populations, providing valuable insights into specific cellular changes in diseases. However, such strategy imposed challenges as cells of interests are often available in limited quantities and quality of RNA may be compromised during long periods of time spent on collection of cells and extraction of total RNA; therefore, it is crucial that protocols for sample preparation should be optimised according to different cell populations.


RESULTS
We made several modifications to existing protocols to improve the total RNA yield and integrity for downstream qPCR analyses. A modified condensed hematoxylin and eosin (H&E) staining protocol was developed for the identification of rat renal proximal tubular cells (PTCs). It was then determined that a minimal of eight thousands renal PTCs were required to meet the minimal total RNA yield required for downstream qPCR. RNA integrity was assessed using at every progressive step of sample preparation. Therefore, we decided that the shortened H&E staining, together with microdissection should be performed consecutively within twenty minutes for good quality for gene expression analysis. These modified protocols were later applied on six individual rat samples. A panel of twenty rat renal drug transporters and five housekeeping genes showed Ct values below thirty-five, confirming the expression levels of these drug transporters can be detected.


CONCLUSIONS
We had successfully optimized the protocols to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR. This protocol may be suitable for researchers who are interested in employing similar applications for gene expression studies.
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, MD11 10 Medical Drive #05-09, Singapore 117597, Singapore. phcyjy@nus.edu.sg.
Jie Yin
JY
Yee
Lie Michael George
LM
Limenta
Keith
K
Rogers
Susan Mary
SM
Rogers
Vanessa S Y
VS
Tay
Edmund J D
EJ
Lee
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D007687:Kidney Tubules, Proximal / Q000737:chemistry* / Q000166:cytology; D061346:Laser Capture Microdissection; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D002352:Carrier Proteins; D018076:DNA, Complementary; D012313:RNA