Introduction
High-quality RNA is required for many molecular biology techniques. The isolation and use of purified RNA in the laboratory is complicated by the fact that, chemically and biologically, RNA is significantly more labile than DNA. The choice and optimization of RNA purification methods are important for successful isolation of high-quality RNA necessary for consistent performance of downstream applications. The Maxwell® 16 purification system combines compact instrumentation, prefilled reagent cartridges and optimized automated methods to maximize performance and flexibility while minimizing hands-on time required for DNA, RNA, and protein purification. The Maxwell®16 LEV simplyRNA Blood Kit is designed for isolation of total RNA from fresh whole blood collected in EDTA tubes. The simplyRNA blood procedure involves only minimal sample handling before automated purification on the Maxwell® 16 Instrument. This kit is designed to isolate high-quality RNA that can be used in many downstream RNA applications. In this study we isolated RNA using the recommended 2.5ml of blood input and optimized the protocol for isolation of more RNA using larger blood input volumes. The quality of the RNA was evaluated using absorbance, gel electrophoresis, bioanalyzer and RT-qPCR analyses.
Methods
RNA isolation. RNA was isolated from blood drawn in EDTA tubes (pooled from three normal healthy donors) using the Maxwell® 16 Instrument with the Maxwell® 16 LEV simplyRNA Blood Kit (Cat.# AS1310). The Maxwell® 16 Instrument (Cat.# AS3000) was fitted with the Maxwell® 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor (Cat.# SP1070), and firmware version 1.40 was installed. Instructions in the Maxwell® 16 LEV simplyRNA Blood Kit Technical Manual #TM372 were followed for isolating RNA using the “Standard” protocol. Several modifications were made to the protocol when increasing the blood input volume above 2.5ml:
- Scale the amount of Cell Lysis Solution proportionately to maintain a ratio of 3:1 Cell Lysis Solution to blood (e.g., for 5ml of blood add 15ml of Cell Lysis Solution [Cat.# A7933]) .
- Increase Homogenization Solution volume from 200µl to 300µl.
- Increase Lysis Buffer volume from 200µl to 300µl.
- Increase Proteinase K volume from 25µl to 50µl (Cat.# MC5008 or V3021).
- Highly viscous samples (8ml of blood) require either:
- Needle Shearing: After addition of Homogenization Solution, Lysis Buffer, and Proteinase K, pass lysate through a 2-gauge needle 5–6 times.
- DNase I Treatment: After resuspension of cells in Homogenization Solution, add 20µl of DNase I, incubate for 5 minutes at room temperature, then proceed with Lysis Buffer and Proteinase K additions.
Analysis of Isolated RNA
Concentration/Yield. RNA isolated using the Maxwell® 16 LEV simplyRNA Blood Kit was analyzed for concentration using both absorbance (NanoDrop®-1000 instrument) and fluorescence (QuantiFluor™ RNA System, Cat.# E3310). The QuantiFluor™ Dye Systems Technical Manual #TM346 was followed to process samples and standards for RNA quantification. The GloMax®-Multi+ Detection System was used to measure fluorescence (replicate of 3), and concentrations were determined by fitting results to a standard curve. RNA samples were diluted 1:200 before adding the QuantiFluor™ RNA Dye.
RNA Integrity. To determine integrity of the isolated RNA, samples were analyzed using gel electrophoresis and bioanalyzer. A total of 4µl of sample was mixed with 4µl of Lonza Formaldehyde loading dye (Lonza Cat.# 50571) and 2µl of Ethidium Bromide Solution (Cat.# H5041, diluted 1:100), and the mixture was heated to 70°C for 2 minutes. Samples were loaded onto a 1.2% agarose gel and run at 100V for 30 minutes. The gel was imaged using a Bio-Rad XR imager. Agilent RNA 6000 Nano Kit (Cat.# 5067-1511) was used to analyze the RNA samples on an Agilent 2100 bioanalyzer. One microliter of sample was added to each well of the RNA chip.
Amplification. The GoTaq® 1-Step RT-qPCR System (Cat.# A6020) was used to reverse transcribe and amplify samples following the protocol provided in the GoTaq® 1-Step RT-qPCR System Technical Manual (TM355). Two experiments were performed, the first using HPRT1 RNA-specific primers and the second using GAPDH DNA-specific primers. Primer concentrations were 100nM in 50µl reactions. RNA samples were diluted 1:20 into water, and 10µl was added per reaction. One microliter of Plexor® HY Male Genomic DNA Standard (Part # DD149A, 50ng/µl) was used as a positive control for the GAPDH primer experiment. Both no-template and no-RT controls were prepared for both RT-qPCR experiments.
Results
To test the scalability of the Maxwell® 16 LEV simplyRNA Blood Kit, RNA was isolated from 2.5, 5 and 8ml of input blood. One EDTA tube contains approximately 8ml of whole blood. When increasing the blood input, the Cell Lysis Solution amount also was scaled proportionately to maintain a 3:1 ratio of Cell Lysis Solution to blood. To ensure efficient nucleic acid release from 5 and 8ml of blood, Homogenization Solution, Lysis Buffer and Proteinase K were scaled as described in the methods section.
Purified RNA samples were first analyzed by absorbance using a NanoDrop®-1000 instrument (Figure 1). The average RNA yield was 5.0 ± 0.2µg using the recommended 2.5ml of blood. Doubling the blood input to 5ml resulted in nearly a doubling of yield to 9.1 ± 1.7µg. Purity ratios (A260/280 and A260/230) for both 2.5ml and 5.0ml blood input samples were >2.0, indicating high purity. Initial experiments increasing blood input to 8ml resulted in multiple problems, including highly variable and significantly reduced yields (data not shown). These samples were highly viscous, and particles were trapped in early wells of the cartridge, thus reducing RNA recovery efficiency.
In an attempt to reduce the viscosity of the 8ml blood lysates, two separate modifications were tested. In one set of samples, the lysate was passed through a needle 5–6 times to shear genomic DNA and reduce viscosity immediately before adding the sample to well #1 of the cartridge. In a separate set of samples, a DNase I incubation step was added to degrade the genomic DNA before adding the sample to the cartridge. RNA was isolated from both sets of samples processed using these modified protocols. Both protocols resulted in consistent yields, with the needle shearing method giving slightly better yields (Figure 1). The purity ratios were above 2.0 , and there was no indication of excessive particle loss in the early wells. RNA from both modified methods were taken through additional analyses.
RNA concentrations also were determined using the QuantiFluor™ RNA System. RNA samples were diluted as described in the methods section, mixed with QuantiFluor™ RNA Dye, and fluorescence was detected using the GloMax® Multi+ Detection System. The data points were fit to a standard curve to determine the RNA concentrations. With the modified kit protocols, increasing blood input resulted in a proportional increase in RNA yield (Figure 2).
Samples also were analyzed on a 1.2% agarose gel and visualized by ethidium bromide staining (Figure 3). A total volume of 4µl of samples were loaded onto the gel. The 2.5ml and 5.0ml blood input samples had prominent 28s and 18s rRNA bands and even show faint 5s rRNA bands. Samples appear to be void of gDNA. All of the samples processed with DNA shearing using a needle or DNase I treatment had prominent 28s and 18s rRNA bands as well, although replicate 5 of the DNase-treated samples showed some RNA degradation.
A bioanalyzer was used to determine RNA integrity numbers (RIN). RIN values are used as a measure of RNA quality, with numbers above 8 representing good quality RNA
(1)
. Samples were analyzed using Agilent Nano 6000 RNA chips on a 2100 bioanalyzer. The 2.5ml and 5ml blood input samples had RIN values >8.7, indicating high-quality RNA (Figure 4). When reducing the viscosity with needle shearing or upfront DNase I treatment, the RIN values were 9.0 ± 0.5 and 8.4 ± 1.7, respectively. Replicate 5 for the DNase I-treated protocol had high degradation with a RIN value of 5.3.
To determine if the purified RNA samples could be reverse-transcribed and amplified, samples were assayed using the GoTaq® 1-Step RT-qPCR System (Figure 5). Samples were diluted 1:20, and 10µl was added to a 50µl reaction. Two sets of primers were used, one specific for RNA (HRPT1) and one for DNA (GAPDH). No-template controls (NTC) and no-reverse-transcriptase controls (–RT) also were added to the reactions. For the RNA-specific primers, NTC and –RT reactions resulted in no detectable Ct values. Ct values for the RNA samples with 2.5ml of blood input resulted in an average Ct value of 34, whereas, Ct values for 5ml and 8ml were 33 and 32, respectively. The lower Ct values indicate an increase in RNA recovered and that the RNA was amplifiable. Using GAPDH primers with –RT as a measure of genomic DNA contamination, minimal (Ct>39) or no product was detected for all RNA samples. This indicates there is little to no DNA copurifying with the RNA.
Table 2. RT-qPCR analysis of RNA samples purified using the Maxwell® 16 LEV simplyRNA Blood Kit. RNA was isolated from 2.5, 5 and 8ml of blood using the Maxwell® 16 LEV simplyRNA Blood Kit. We followed the standard protocol outlined in the the Maxwell® 16 LEV simplyRNA Blood Kit Technical Manual #TM372. The “Modified” protocol increased the volume of reagents. The “Modified + Needle Shearing” and “Modified + DNase I” protocols added preprocessing steps to reduce viscosity of lysates before addition to the Maxwell® 16 cartridge. This chart represents two separate experiments: one with RNA-specific primers and one with DNA specific primers. Ct values are displayed. N/D represents that a Ct value could not be determined. The DNA control was genomic DNA standard.
Conclusions
The Maxwell® 16 simplyRNA LEV Blood Kit can be used to isolate high-quality RNA from greater than 2.5ml of blood. RNA quality was assessed by absorbance purity ratios, gel electrophoresis and bioanalyzer. All samples performed in RT-qPCR and had little to no detectable genomic DNA carryover. As blood input is increased, RNA yields increased proportionally. When increasing above 5ml of blood, preprocessing was required to reduce viscosity and produce higher yields and quality with more consistency. Preprocessing by needle shearing gave the best results, but a short DNase digest also may be used. Processing larger blood volumes (>2.5ml) will require purchase of additional Cell Lysis Solution (Cat.# A7933) and Proteinase K (Cat.# MC5008 or V3021).