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
Recover High Quality Total RNA from Blood
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 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.