mRNA extracted from frozen buffy coat samples stored long term in tubes with no RNA preservative shows promise for downstream sequencing analyses

Abstract

Transcriptomics is an important OMICs method that is often unavailable in biobank research. Frozen blood samples are routinely collected and stored in medical biobanks, but transcriptional studies have been limited due to technical difficulties of extracting high-quality RNA from blood frozen in standard tubes (without RNA preservatives). We aimed to determine whether biobanked buffy coat samples stored at -80°C for up to 23 years could be successfully used for mRNA sequencing. We used a CryoXtract CXT 350 to remove frozen sample cores, which were immersed in RNA preservative during thawing prior to RNA extraction. RNA sequencing was then performed on extractions from pooled samples as well as from 23 buffy coat samples from prospective colorectal cancer cases and 23 matched controls included in the population-based, prospective Northern Sweden Health and Disease Study (NSHDS). For all samples, two library preparation methods were used (Illumina TruSeq Stranded mRNA poly-A selection and Illumina Stranded Total RNA with Ribo-Zero Globin). RNA yields of over 1 µg were obtained from the majority of NSHDS samples (mean = 2.57 µg), and over 92% of samples had RIN values of ≥ 6, indicating suitability for downstream analyses. In conclusion, we developed a method for successfully extracting and sequencing high-quality mRNA from frozen buffy coat samples stored long term in tubes with no RNA preservative.

Fig 1. Flowchart depicting the origin of all sequenced samples.

Fig 2. (A) Individual RNA yields from the training samples (n = 80) and the NSHDS samples (n = 56) (total μg from each sample). (B) Variations in RIN values from the training set and the NSHDS samples. (C and D) RIN curves of an individual sample within each sample group which are also representative of the mean RIN values for the training set (C) and NSHDS samples (D).

Fig 3. (A) RIN values of RNA extracted from pooled buffy coat after different storage and handling conditions.

(B-D) Max RIN curves for fresh (B), frozen (C) and frozen +  cryoextracted (D) pooled buffy coat.