Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2012) 30 P46

BSPED2012 Poster Presentations (1) (66 abstracts)

Extraction of high quality RNA from fresh frozen and formalin fixed paraffin embedded human pancreatic tissues samples of patients with congenital hyperinsulinism for gene expression microarray

Senthil Senniappan , Amanda Heslegrave & Khalid Hussain

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UCL Institute of Child Health, London, UK.


Introduction: The molecular research on rare diseases is limited by the availability of tissue samples that yield good quality RNA. Extraction of RNA from human pancreatic tissues are challenging due the high amount of ribonucleases.

Aim: To compare the quality of RNA from fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) human pancreatic samples of patients with congenital hyperinsulinism (CHI).

Methods: Tissue samples were obtained from children CHI who underwent surgery. RNA was extracted from seven FFPE (four focal and three normal) and eight FF samples (six diffuse and two normal) by standard techniques (TRIzol Reagent). RNA Integrity is assessed by Agilent-derived RNA Integrity Number (RIN) and the presence of intact 18s and 28s bands on the Agilent Bioanalyzer trace. The purity is determined by the optical density ratio (OD 260/280).

Results: The RNA yield from FFPE and FF samples were 327 and 1032 ng/μl respectively. FFPE samples yielded a very low RIN (mean 2.3; range 2–2.4) whilst FF samples yielded high RIN values (mean 7.0; range 5.8–8.6). The mean OD 260/280 ratio for the FFPE samples was 2.16 and that of FF samples was 1.9. All the FF samples had intact 18s and 28s bands whilst it was absent in all of the FFPE samples. The microarray Qc from FF samples revealed a mean pos-v-neg auc value of 0.84 which suggests a good quality data.

Conclusion: A RIN factor greater than 7, OD 260/280 ratio between 1.8 and 2.1 and Intact 18s and 28s bands are markers of good quality RNA that could be used for gene expression microarray. Whilst extracting good quality RNA from human pancreatic samples for microarray studies remain a challenge, FF samples yield better quality RNA than FFPE samples.

Volume 30

40th Meeting of the British Society for Paediatric Endocrinology and Diabetes

British Society for Paediatric Endocrinology and Diabetes 

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