Simultaneous Isolation of DNA, RNA, and Proteins for Genetic, Epigenetic, Transcriptomic, and Proteomic Analysis

Abstract

Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

Fit for Purpose Frozen Tissue Collections by RNA Integrity Number-Based Quality Control Assurance at the Erasmus MC Tissue Bank

“…Some reports describe the simultaneous isolation of RNA, DNA, and proteins. 13,[15][16][17] There seems to be a correlation between RNA, DNA, and protein quality when different sample types such as paraffin embedded tissue and serum are considered. However, this does not say anything about an eventual correlation between RIN value and DNA/ protein quality within one specific sample type group, namely frozen tissue.…”

Correlation of telomere length shortening with promoter methylation profile of p16/Rb and p53/p21 pathways in breast cancer

“…Using the two-way hierarchical cluster analysis, the most variable CpG fragments for each gene were clustered based on pair-wise Euclidean distances and linkage algorithm for all studied samples according to the previously developed method. [19][20][21] For gene clustering, pair-wise similarity metrics were calculated for each gene separately based on methylation ratio of cancer tissues across the adjacent paired normal tissues. The procedure was carried out using the double dendrogram function of the Gene Expression Statistical System for Microarrays (GESS) version 7.1.19 (NCSS, Kaysville, UT, USA).…”

“…17,[19][20][21] Briefly, bisulfite conversion of the target sequence was obtained using the Epitect bisulfite kit (QIAGEN AG, Basel, Switzerland). Primers were designed to cover the promoter regions with the most CpG sites.…”

“…23,24 In this study, we explored the feasibility of using paraffin-embedded tissues for the quantitative assessment of telomere length together with highthroughput methylation quantification on MALDI-TOF silico-chips. 20,21 Before carrying out telomere length analysis, yield of extracted DNA from formalin-fixed paraffin-embedded tissues was quantified using NanoDrop ND-1000 spectrophotometer (Biolab, Mulgrave, Vic, Australia). Median DNA quantities was determined as 37.92 ng/ml.…”

Calcification of vascular smooth muscle cells is induced by secondary calciprotein particles and enhanced by tumor necrosis factor-α

“…PCR amplification was performed using the oligonucleotide primers (Suppl. Table S1 lists primer sequences) SYBR Green PCR Master Mix (Roche, Switzerland) [29]. Melt-curve analysis was performed to confirm the presence of a single specific product and non-template controls run to assess contamination.…”