Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

Abstract

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

Gene Expression Analysis by qPCR in Clinical Kidney Transplantation

“…Alternatively, the cells can be stored in RPMI culture medium and DMSO in liquid nitrogen, using conventional freezing procedures. In this way, RNA is equally well preserved as in RNA later [ 19 ]. RNA can still be extracted from the cells at a later time point, but then the cells need to be thawed fi rst and washed, to get rid of the DMSO.…”

“…Over the last years, microRNAs have gained attention because of their role in disease. Since small RNAs (<200 nucleotides) are for a large part lost with the RNeasy columns [ 19 ], we now are using a kit that is suitable for isolating both small and large RNAs. The yields of conventional (large) mRNA obtained with this kit are equal to those obtained with RNeasy [ 19 ].…”

Polyclonal B cell activation for accurate analysis of pre-existing antigen-specific memory B cells

“…Total RNA was extracted using the NucleoSpin miRNA kit (Bioké, Leiden, the Netherlands), according to the manufacturer's instructions. Complementary DNA synthesis was carried out using SuperScript III reverse transcriptase (Life Technologies, Paisley, UK), as described previously [22]. Polymerase chain reaction (PCR) assays were performed with each of seven forward primers covering the different VH1-7 chains in combination with a fluorophore-labelled reverse primer specific for the constant region of IgM (μ) or IgG (γ).…”

MicroRNAs in veterinary cardiology

“…Despite a promise in veterinary cardiology exists, there are some obstacles to the use of microRNAs in the clinical routine, such as their instability in the sample and a degradation of the molecules, which may affect the results of the expression analyzes. For this, there are several extraction protocols aiming to minimize these effects (EIKMANS et al, 2013).…”