Extraction of total RNA and isolation of mRNA is an intermediate step done prior to its use in further applications. The purpose of this experiment was to determine an efficient way to assess total RNA and mRNA prior to using the sample in a microarray-based study. Twenty hours after Saccharomyces cerevisiae (yeast) was plated, RNA was extracted using Ambion’s RiboPure Yeast Kit. Absorbance readings at 260nm and 280nm were obtained to determine RNA quality and quantity. Another method to determine relative quantity and quality of RNA samples involved different gel electrophoresis protocols, including the use of proprietary (Viagen, Inc.) Superload denaturing buffers and native agarose gels. The presence of bands at 1800 base pairs (bp) and 3000bp representing the 18S and 28S ribosomal RNA subunits confirmed total RNA presence and relative quantity. Electrophoresis of the extracted RNA, treated with denaturing buffers, was the most efficient protocol in verifying the presence of total RNA. In order to determine whether mRNA, which is needed in microarray-based applications, was present within the total RNA sample, New England BioLab’s Protoscript First Strand cDNA Synthesis Kit was used to make cDNA from any mRNA that was present in the sample via Polymerase Chain Reaction (PCR) using reverse transcriptase. PCR was also used to amplify TDH1, a housekeeping gene present in mRNA. The resulting gene was visualized on a gel and found to be present at approximately 200bp as expected.
The purpose of this experiment was to analyze total RNA prior to utilization in a microarray or other gene expression study. The early detection of mRNA ( or absence of it) saves time, energy and resources in the laboratory, giving the scientist the ability to better determine the other sources of error present in the experiment (Brewster et al., 2004). Brewer’s yeast or Saccharomyces cerevisiae was the organism chosen for study. In addition to being a eukaryote, it was selected for its simplicity, rapid growth, low cost, and genomic sequence availability (Sherman, 1998). Different protocols to establish mRNA presence from total RNA, including its quality and quantity were examined to determine the most precise and least labor intensive protocol. Results of this study were used to determine which samples could be used in a microarray-based study of yeast gene expression when exposed to the same environment (Farrell, 2005).
S. cerivisiae (S288C) was grown according to the Genome Consortium for Active Teaching protocol (GCAT, 2005). A loopfull of yeast was placed into 5 mL of liquid media, a 1 mL aliquot of this suspension was placed in 40mL of liquid yeast growth media (GCAT, 2005). Six volumes of this yeast culture (11mL, 11mL, 5mL, 2mL, 1.5 mL, 1.5 mL) were taken for RNA extraction following Ambion’s RiboPure Yeast Kit (Austin, TX, USA).
Absorbance readings of the extracted RNA were obtained at two different time periods: shortly following RNA extraction and four months after extraction. At the first time period, the RNA was quantified and qualitatively assessed by obtaining absorbance readings using the Hewlett Packard UV Visible Spectrophotometer at 260nm and 280 nm (using crystal cuvettes). Four months after the extraction, absorbance readings were repeated using a small volume (100 μL) spectrophotometer (SmartSpec).
RNA quantity was calculated as follows:
A260 x dilution factor x (40 μg/mL/1000)
(RiboPure Manual, 2004). RNA quality was assessed by its absorbance ratio (A260:A280). Pure RNA samples are known to have ratios within the range of 1.8 and 2.1 (RiboPure Manual, 2004).
Electrophoresis of mRNA
Electrophoresis was performed to determine mRNA presence by visualizing bands at 1800bp and 3000bp, representing the 18S and 28S subunits of rRNA, with a visible smear between bands representing mRNA. The HS Reliant Gel System, a pre-packaged gel treated with GelStar stain was used for electrophoresis (Cambrex BioScience, Baltimore, MD, USA). 5 μL RNA with 1 μL of loading dye was run through the gel for one hour at 100 V and 130 mA. DNA ladders (1 kB and 50 bp) were also run for comparison of molecular weight.
Polymerase Chain Reactions
The extracted RNA was used as a template to produce cDNA via RT-PCR, using a kit obtained from New England Bio Labs (Protoscript First Strand cDNA Synthesis Kit). To take advantage of the PolyA tail found at the 3’end of mRNA, oligo-DT primers, reverse transcriptase, and dNTPs were used to make cDNA. Since cDNA cannot be directly visualized in an agarose gel, TDH1, a “house-keeping”gene present in all strands of mRNA was specifically targeted by adding specially designed primers prior to amplification by PCR. This PCR protocol involved: 25 μL deionized water, 1 μL 25 mM MgCl2, 1 μL of forward and reverse primer each at 5mM concentration, 13 μL Quiagen Master Mix (Quaigen, Valencia, CA, USA) and 5 μL cDNA template sample. The thermocycler was set to 20 cycles of 94°C for 15 seconds, 58°C for 15 seconds and 74°C for 30 seconds. Following PCR 1 μL loading dye, 1μL (1:1000) Sybr dye and 5μL of amplified TDH1 gene was electrophoresed on a 1.0% TBE (native) agarose gel for approximately 1 hour at 100V and 130 mA.
Four months after the original extraction of RNA, another protocol, was examined to determine an efficient method that enables RNA separation in native gels (Gregg et. al, 2004). The method required the use of a denaturing buffer, Superload (ViaGen, Austin, TX, USA) in addition to a small amount of RNA (1 μg). For each sample, 2 μL of the total RNA was used, in addition to 1 μL buffer A, 3 μL buffer B, 1 μLSybr Gold (1:1000) and 1 μL loading dye (Superload Gregg et al., 2004). The gel was run at 100 volts for one hour and 30 minutes on a 1.5% TBE agarose gel.
Table 1: Obtained absorbance of total RNA samples after extraction, on September 30, 2004.
|Total RNA Sample||Amount of yeast culture originally taken (mL)||Dilution factor
(RNA: deionized water)
|A280 (Absorbance units)||Ratio (A260:A280) Using 1000μL||RNA Quantity (μg/μL)**|
Table 2: Absorbance on remains of total RNA samples, performed on January 20, 2005.
|Amount of yeast
Figure 1: RNA electrophoresed using ready made HS Reliant Gel System.
Figure 2: RNA samples electrophoresed approximately four months after RNA extraction, using Superload Denaturing Gel Loading Buffer (Viagen) with 4μL of total RNA. The top band at approximately 3000bp was darker than the second band at 1800bp. A minimal amount of RNA (<2μL), from sample five, was available for this protocol.
Figure 3: Electrophoreses of cDNA, after amplification of the TDH1 gene. cDNA produced via RT-PCR from RNA samples two and five displayed smeary a light, smeary band at approximately 200bp.
Figure 4: Yellow spots on a microarray appeared after two specimens of cDNA, each labeled with their own Cy-3 or Cy-5 dye, were hybridized successfully. The cDNA was produced by RT-PCR from mRNA, sample 2.
RNA Samples 2 and 3 were of the highest quality after extraction, with absorbance ratios (A260:A280) of 1.8, while sample 5 had a ratio of 1.3. Samples 1 and 2 had RNA concentrations: 3.8 μg/μL and 2.3 μg/μL, respectively measured on 9/30/04 (Table 1).
All absorbance ratios were between 1.8-2.1. when the remains of the original, extracted RNA, were measured using a more accurate, small volume spectrophotometer (on 1/20/05). Most of the samples, with the exception of sample 3 had a lower RNA concentration (Table 2).
RNA band Visualization
The HS Reliant Gel System allowed the two ribosomal subunits to be visualized (1500bp and 3000 bp) along with smears representing DNA fragments of low molecular weight (Fig. 1). The gel prepared with 2 μL of total RNA, treated with Superload Denaturing Buffer allowed the visualization of two distinct bands at approximately 2500bp and 1500bp. The 2500bp band was approximately one and a half times brighter than the 1500 bp band, with a light smear present in between the bands. Samples 1, 2 and 3 (second and third lanes from the left) clearly demonstrated bands; however, samples 4, 5 and 6 had faint bands (of the same molecular weight) that were not visualized by the camera (Fig. 2).
TDH 1 Visualization
Only samples two and five fluoresced bands at approximately 200 bp indicating the presence of the TDH1 gene (Fig. 3). These samples were chosen for hybridization onto a microarray.
Samples two and five were fluorescently tagged and hybridized onto a microarray (Farrell, 2005). Figure 4 demonstrates the microarray produced, originally from sample 2, containing yellow spots.
Spectrophotometry lacks the ability to definitively confirm mRNA presence. Tables 1 and 2 demonstrate that apparent differences in quality and quantity within the specimens, and between the two different time periods. It should be noted that different dilution factors and solvents were used at the different measurement times (deionized water or TBE buffer). The different dilution factors were included in the calculations of RNA concentration. Appropriate measures were also taken to use the same solvent as the blank for each set of readings.
When RNA concentrations were measured the second time they were mostly lower, while absorbance ratios were found to be higher. These differences are likely due to the higher precision of the spectrophotometer that was used second since measurements were made using a very small volume (100 μL instead of 2000 μL). Additionally, results may have been affected by the samples being old, having been exposed to repeated freeze-thaw sessions, possibly becoming diluted from improper mixing, and inadequate temperature for storage at -20°C. Samples should be stored at -80°C for longer than two months (RiboPure Protocol).
RNA Bands Visualization
mRNA is needed for microarray labeling (Farrell, 2005). Concerns arise regarding the integrity of native agarose gels in providing the results necessary to confirm mRNA presence. Other protocols recommend the use of a denaturing gel to eliminate the possibility of degraded RNA visualization allowing the true visualization of mRNA. On the other hand, denaturing gels, require carcinogenic materials, and additional time.
Smears in the agarose gel indicate that there were substances of a low molecular weight present. These bands were likely “products of DNase digestion (Ambion, 2005).” Because of the smears present, the possibility of secondary RNA structure interfering with banding patterns and the large quantity of RNA sample used, this method was not ideal (Fig. 1).
RNA treatment with Superload denaturing buffer, (ViaGen, Austin, TX, USA), allowed RNA band visualization without the low molecular weight smears and eliminated the possibility of secondary RNA structure interfering with banding patterns. This gel was run approximately four months after the original extractions, therefore, RNA degradation may have occurred. This time delay may also have lead to the bands being visualized at slightly smaller than expected sizes. The absence of bands for Samples 4, 5 and 6 were due to the use of <2μL of RNA, because of its inavailability after previous protocols. It is also believed that because the ladders were not treated with denaturing buffer, the actual molecular weight determined by comparison to the ladder was affected (Fig. 2).
The bands at approximately 200bp provided results confirming the original presence of mRNA in the total RNA samples. Although this step proved to be helpful in our experiment, the amplification of the TDH1 housekeeping gene was a lateral step in the experiment, preventing the course of progression; therefore this method was not ideal if RNA presence could be reliably determined from gel visualization.
The use of a denaturing buffer, in addition to spectrophotometry was determined to be the most efficient method for RNA assessment. Since microarrays and other gene studies provide vast amounts of genomic information, it is important to limit experimental variables that could affect the results. Since the denaturing buffer required a small volume of RNA, and only requires a small, additional step to treat samples with denaturing buffers, this test can be run directly after extracting, streamlining the process. This allows microarray hybridization to occur sooner, decreasing chances of DNAse or RNase contamination. This protocol also facilitated cost effectiveness since expensive reagents will not be wasted on samples that may not have RNA present, such as those used n PCR.
The scientist would like to thank Dr. Emily Schmitt for her advice on the research, and the following for their contributions to the research: Dr. Pomeroy for providing quartz cuvettes; Dr. Kozarov for the use of her spectrophotometer; Dr. Edkahl for the yeast and the TDH1 visualization procedures; Mary Lee Ledbetter, Holy Cross College; GCAT and Maria Farell for her input and assistance. Additionally, the researchers were grateful to the following companies for their donations: ViaGen, Dr. Brian Bruner and Ambion, Ginger Zara.
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