Mammalian whole blood is a widely used tissue for molecular assays due to abundant genetic information as well as ease and simplicity of sample collection. RNA isolation from whole blood is challenging, but Ambion’s MagMAX™-96 Blood RNA Isolation Kit uses strong denaturing lysis conditions and proteases to effectively inactivate ribonucleases and remove blood proteins.
MagMAX-96 Blood Technology
For RNA purification, Ambion’s magnetic bead-based MagMAX technology provides a fast, simple protocol that eliminates the use of organic solvents as well as problems associated with glass fiber filter-based methods (e.g., filter clogging and inconsistent RNA yield). Whole blood (and milk) samples can be processed manually using multichannel pipettes or adapted for automation to recover total and viral RNA of high yield, quality, and purity. The extracted RNA is ideal for downstream molecular diagnostics and gene expression profiling using quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
Recover High Quality Total RNA from Blood
High quality total RNA from human whole blood (fresh or stored at 4°C) can be easily and efficiently recovered using MagMAX-96 Blood. For example, total RNA (8 replicates from multiple donors) was isolated using the standard MagMAX-96 Blood protocol, which involves tissue/cell lysis and magnetic bead sample capture followed by TURBO DNase treatment. High quality, intact RNA with 28S/18S rRNA ratios >0.9 (Agilent® 2100 bioanalyzer) was obtained (Figure 1). RNA yield was determined by A260 with an average range of 0.31–0.52 µg total RNA from all samples (50 µl each).
Figure 1. MagMAX™-96 Blood Consistently Recovers Intact Total RNA from Fresh Blood and Blood Stored at 4ºC.
Human whole blood from four different donors was collected into EDTA–Vacutainer® tubes; aliquots were stored at 4ºC for 24 hr. Fresh whole blood was collected from the same donors the following day for concurrent RNA isolation with the samples stored at 4ºC. Total RNA was isolated from 50 µl blood from multiple donors in 8 replicates using the MagMAX-96 Blood Kit, and total RNA (~50 ng) from representative samples was analyzed on an Agilent® 2100 bioanalyzer (A). RNA yield was determined by A260 measurement using the NanoDrop® ND-1000A Spectrophotometer (B).
Genomic DNA contamination was determined by one-step qRT-PCR targeting hTBP (human TATA binding protein) and RPII (RNA polymerase II) mRNAs [(+)RT reactions] and genes [(-)RT reactions]. Total RNA purified from all samples contained minimal genomic DNA contamination as assessed by delta-Ct values from qRT-PCR (Figure 2). Similar (+)RT Ct values of qRT-PCR (hTBP and RPII) using RNA isolated from fresh blood and blood stored at 4°C were obtained.
Figure 2. Comparison of Total RNA Isolated from Fresh Blood and Blood Stored at 4°C in qRT-PCR Assays.
Total RNA (~25 ng) was isolated with the MagMAX™-96 Blood Kit and was used in qRT-PCR targeting the TATA Binding Protein (TBP, panel A) and RNA Polymerase II (RPII, panel B) mRNA [(+)RT reactions] and genes [(–)RT reactions]. Minimal genomic DNA contamination was revealed by the Ct values of (+)RT and (–)RT reactions. Similar total RNA yield and quality were obtained from fresh and blood stored at 4°C (for 24 hr) as demonstrated by similar (+)RT Ct values.
RNA isolated from blood stored at -80°C had similar yields but lower integrity than samples from fresh blood or blood stored at 4°C (data not shown). RNA degradation may result from hemolysis during thawing; however, the RNA can still be used for successful qRT-PCR yielding valuable gene expression profiling.
High Throughput Isolation of Blood RNA for Veterinary Molecular Diagnosis
The ability to directly use whole blood for total and viral RNA isolation decreases cross contamination and saves time and resources. This new kit is designed for high throughput viral and total RNA isolation of 50 µl samples in a 96 well plate. The MagMAX-96 Blood RNA Isolation Kit has been used successfully for total and viral RNA from bovine (Figure 3), porcine, and ovine whole blood, as well as bovine milk samples, allowing high throughput viral, veterinary molecular diagnosis.
Figure 3. MagMAX™-96 Blood Is Easily Adaptable for Automation. Bovine viral diarrhea virus (BVDV) infection is a major problem of worldwide distribution that results in economic losses for the beef and dairy industries. Blood and milk are ideal sample sources for BVDV RNA isolation due to the simplicity and ease of sample collection. BVDV positive and negative bovine blood samples were aliquoted (50 µl) into separate wells of a 96 well plate and processed using the MagMAX-96 Blood protocol on a Biomek® 2000 (Beckman Coulter). Control RNA was spiked into each sample well at equal copy numbers to monitor sample processing consistency of the 96 well plate. The purified RNA was subsequently used for qRT-PCR targeting BVDV RNA and control RNA. BVDV target amplification was highly consistent: average Ct=21.4 ± 0.5. Control RNA amplification was equivalent (average Ct=28.3 ± 0.4) across the 96 samples. No cross contamination was observed. Processing a full plate of samples requires ~1 hr.
The MagMAX-96 Blood protocol permits rapid (<1 hr) total and viral RNA isolation from blood samples (up to 50 µl) and is also applicable for viral RNA isolation from milk samples. The RNA is eluted in a low salt buffer (20–50 µl) and can be directly used for qRT-PCR.
Angela Burrell, Quoc Hoang, Roy Chris Willis, WeiWei Xu, Mangkey Bounpheng, Xingwang Fang • Ambion, Inc.