BioLiqX RNA Isolation Kit includes all the components required to isolate total RNA from liquid samples including blood plasma and serum. The method is based on prior lysis of a sample with phenol-containing BioliqX Lysis Buffer R, subsequent phase separation and spin-column purification of RNA. The protocol efficiently denatures protein-rich biological fluids and ensures the complete inactivation of RNases. Furthermore, the optimized composition of the BioliqX Lysis Buffer R enables superior recovery of highly diluted ultra-short RNA fragments and almost complete removal of the DNA during organic extraction. In addition, optimized washing buffers ensure the absence of enzyme-inhibiting traces of phenol in the RNA eluates. Finally, ultra-clean spin columns secure the absence of contaminating RNA and allow sample elution into the minimal volume of as little as 10 μL. The whole procedure can be completed within approximately 1.5 hours and requires a hands-on time between 10-30 minutes depending on the number of samples.
The kit is delivered in a compact carton box containing 50x Spin Columns, 50x Collection Tubes, 50x Nuclease-Free Tubes, RNAse-Free Water as well as ethanol-free concentrates of Washing Buffer 1 and Washing Buffer 2. The phenol-containing BioliqX Lysis Buffer R is supplied in a separate dark glass bottle.
The following components are not included and have to be supplied by the user:
Safe-Lock 2 mL microcentrifuge tubes for the initial sample lysis (e.g. from Eppendorf AG)
The volume of the initial sample can be custom but will be limited by the volume of the microcentrifuge tube used for the initial sample lysis. While the kit is designed for 400 μL sample volume in a 2.0 mL microcentrifuge tube, various other inputs can be used (e.g. 200 μL of sample in a 1.5 mL microcentrifuge tube). Importantly, the final sample volume should be at least 200 μL to minimize the loss of the aqueous upper phase after organic extraction. We highly recommend using a synthetic spike-in cel-miR-39 (or similar small ssRNA) control for (1) assessing the efficacy of the RNA isolation between samples, and (2) normalization of miRNAs (or other ssRNAs) expression in biological fluids after downstream RT-qPCR. The synthetic spike-in cel-miR-39 control is also included in BioLiqX cf-RNA Isolation QC Kit which is highly recommended for assessing the isolation efficacy of cell-free RNA.
The procedure is suitable for use with biological fluids and other liquid samples containing citrate or EDTA. Plasma samples prepared from blood collected on heparin should not be used as heparin significantly interferes with multiple downstream applications including RNA sequencing and RT-qPCR
The RNA isolation procedure includes three general steps. First, the sample is lysed in 3 volumes of BioliqX Lysis Buffer R and thoroughly mixed. The lysate is then separated into the upper aqueous and the lower organic phases by adding 0.11 volumes of chloroform and high-speed centrifugation. The proteinous solid interphase can be also formed after centrifugation in some samples. The upper phase containing the RNA is collected for the downstream procedure while the lower phase and the interphase are discarded. In the second step, the 1.5 volumes of 100% ethanol are added to the upper phase to facilitate the binding of RNA to the silica support. The sample is then applied to a corresponding spin column, where the RNA binds to the membrane while other contaminants remain in the flow-through. In the final step, the spin column is washed sequentially with Washing Buffer 1, Washing Buffer 2 and 80% ethanol, and the high-quality RNA is eluted in the appropriate volume of RNase-Free water.
Total RNA was isolated from 0.4 mL of RNAse-free water (mock isolation) with either BioLiqX RNA Isolation kit (MB) or competing kit (MC) according to the manufacturers' protocols (final elution volume - 50 µL). Subsequently, 8 µL of the mock isolates, synthetic cel-miR-39 and RNAse-free water were used as inputs for NGS libraries preparation with BioLiqX Small RNA-seq Kit using 15 PCR cycles pre-amplification. The left figure shows 3% agarose gel electropherogram of the final NGS libraries after the final pre-amplification step.
Total RNA was isolated from 0.4 mL of cell debris-free human blood plasma with either BioLiqX RNA Isolation kit (B1, B2), competing phenol-free RNA isolation kit (C1, C2) and competing phenol-based kit (C3) according to the manufacturers' protocols. Synthetic cel-miR-39 DNA (DC39) served as a positive control for DNA-seq library preparation with a ligation-free method CATS as described by [Turchinovich et al, 2014]. The samples were eluted in 50 µL of RNAse-free water, and 8 µL was used as inputs for the library preparation with 15 PCR cycles pre-amplification. The left figure shows 3% agarose gel electropherogram of the final DNA libraries. Note, co-isolation of significant amounts of cell-free DNA is
Total RNA was isolated from 0.4 mL of cell debris-free human blood plasma with either BioLiqX RNA Isolation kit or competing phenol-free RNA isolation kit according to the manufacturers' protocols. Synthetic cel-miR-39 (0.5 pg) was spiked-in after the initial lysis step in each sample as a reference for RNA isolation efficacy. Upon elution in 50 µL of RNAse-free water, 5 µL of RNA were taken as inputs for RT-qPCR assessment of endogenous hsa-miR-16 and hsa-miR-21 as well as spiked cel-miR-39 with BioLiqX cf-RNA Isolation QC Kit. The qPCR was performed using LightCycler 480 Real-Time PCR System (Roche). Raw threshold cycle (Ct) values were determined by the second derivative max method. Each bar represents the mean (SD) of three independent RNA isolations.