ArticleBMC research notes2014
Ensuring good quality RNA for quantitative real-time PCR isolated from renal proximal tubular cells using laser capture microdissection.
Article in BMC research notes, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers, 1 of them a synthesis that pooled it.
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Who cites it
9 citing papers in PubMed, 1 synthesis or guideline pooled it, 19 citations in OpenAlex.
- Molecular analysis of H&E- and Papanicolau-stained samples-systematic review.Histochemistry and cell biology · 2020Pooled it
- Effects of proximal tubule-specific ATRAP enhancement on hypertension in a remnant kidney chronic kidney disease model of mice.Scientific reports · 2025Article
- A Critical Issue in Lung Cancer Cytology and Small Biopsies: DNA and RNA Extraction from Archival Stained Slides for Biomarker Detection through Real Time PCR and NGS-The Experience in Pathological Anatomy Unit.Diagnostics (Basel, Switzerland) · 2023Article
- Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping.International journal of molecular sciences · 2022Article
- Effects of ATRAP in Renal Proximal Tubules on Angiotensin-Dependent Hypertension.Journal of the American Heart Association · 2019Article
- Laser capture microdissection for transcriptomic profiles in human skin biopsies.BMC molecular biology · 2018Article
- Laser capture microdissection protocol for gene expression analysis in the brain.Histochemistry and cell biology · 2017Article
- Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing.BMC genomics · 2016Article
- Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis.PloS one · 2015Article
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundIn 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.
findingsWe 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.
conclusionsWe 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.
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