Ensuring good quality RNA for quantitative real-time PCR isolated from renal proximal tubular cells using laser capture microdissection

Abstract

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.

Findings: 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.

Figure 1

Identification of rat renal PTCs via modified H&E staining protocol. The shortened H&E staining procedure did not compromise on the identification of rat renal PTCs. The rat renal PTCs were recognized by their distinct brush border membranes and were circled in red. Renal distal tubular cells and glomerulus were outlined in yellow and blue respectively.

Figure 2

Total RNA concentrations from different number of renal cells. Total RNA concentrations were measured using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit. It was observed that 3000 renal cells yielded a total RNA concentration too low to be detected while 8000 and 18000 renal cells produced similar total RNA yields.

Figure 3

Effects of DNase I treatment modifications on Ct values. It was shown that Ct value of gDNA control increased (i.e. gene expression reduced) after modified DNase I treatment was applied. On the other hand, the modified DNase I treatment did not affect the Ct values of positive plate control.

Figure 4

Gene expression profiles of renal drug transporters located on rat renal PTCs. All twenty renal drug transporters and five housekeeping genes (ACTB, GAPDH, PPIA, PGK1 and 18SrRNA) had Ct values lower than 35 which confirmed the expression of these selected drug transporters on rat renal PTCs.