SHORT REPORT

A reliable method for total RNA extraction from frozen human bone marrow samples taken at diagnosis of acute leukaemia

D Barbaric¹, L Dalla-Pozza², J A Byrne³

¹ Molecular Oncology Laboratory, Oncology Research Unit, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead 2145, NSW, Australia
² Oncology Department, The Children’s Hospital at Westmead
³ The University of Sydney Department of Paediatrics and Child Health, The Children’s Hospital at Westmead

Correspondence to:
Correspondence to:
Dr J A Byrne, Molecular Oncology Laboratory, Oncology Research Unit, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead 2145, NSW, Australia;
JennifeB{at}chw.edu.au

This report describes a newly developed method using Trizol LS^® reagent that can reliably extract high quality total RNA from frozen human leukaemic bone marrow samples. Extraction of total RNA from 71 frozen leukaemic bone marrow samples obtained at the time of diagnosis produced a median yield of 145 µg/ml leukaemic bone marrow. Total RNA samples could be reverse transcribed into cDNA and used successfully in the reverse transcription polymerase chain reaction amplification of B2M transcripts in 68 of 71 cases. A multivariate linear regression analysis revealed that significant predictors of RNA yield were both sample volume (< 1 ml v > 1 ml; p = 0.003) and peripheral blood white cell count (< 5 x 10⁹ v 5 x 10⁹ white blood cells/litre; p = 0.011). The percentage of blasts present, leukaemia subtype, and sample storage period at -80°C (up to 945 days) were not predictors of total RNA yield. This method of total RNA extraction should be of interest to diagnostic and research staff using frozen bone marrow samples for molecular analyses. Similarly, the lack of association between sample storage period at -80°C and total RNA yield should be of interest to the administrators of tumour banks housing frozen bone marrow samples.

Keywords: RNA extraction; RNA yield; bone marrow; leukaemia; tumour bank

Abbreviations: ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; B2M, ß₂ microglobulin; BM, bone marrow; RT-PCR, reverse transcription polymerase chain reaction; WCC, white blood cell count

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