Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank

Abstract

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

“…Critical variables that influenced the quality of the RNA also included the conditions of the tissue, or the impact of degradation, during processing steps. The vulnerability of RNA in rat ILMs contradicts findings that characterize skeletal muscle mRNA as highly stable relative to the liver and brain 10,11 . These evaluations suggested that variable RNA stability across species, tissue types, and organ layers may require modified extraction strategies according to the specimen and sample size.…”

“…2A). The applied RNase inhibiting reagent (RNAlater), which hinders RNase activity through precipitation and metal chelation 10 , significantly increased the RIN in the overall model (P < 0.0001, Fig. 2A, Table 1).…”

“…Pathological investigation of laryngeal muscle tension disorders is particularly favorable in the rat, a well-studied model of RLN injury and vocal fold paralysis 8 . Unfortunately, ILMs have not been amenable to high-throughput assessments as readily as other soft tissue types due to technical and biological challenges 10 . The impact of these challenges on the RNA extracted from skeletal muscles, including the ILMs, is consistent across species 11 .…”

An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

“…Critical variables that in uenced the quality of the RNA also included the conditions of the tissue, or the impact of degradation, during processing steps. The vulnerability of RNA in rat ILMs contradicts ndings that characterize skeletal muscle mRNA as highly stable relative to the liver and brain 10,11 .…”

“…Pathological investigation of laryngeal muscle tension disorders is particularly favorable in the rat, a well-studied model of RLN injury and vocal fold paralysis 8 . Unfortunately, ILMs have not been amenable to high-throughput assessments as readily as other soft tissue types due to technical and biological challenges 10 . The impact of these challenges on the RNA extracted from skeletal muscles, including the ILMs, is consistent across species 11 .…”