Yeast Culture
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).
Spectrophotometry
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.
Denaturing Gel
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) |
A260
(Absorbance units) |
A280 (Absorbance units) |
Ratio (A260:A280) Using 1000μL |
RNA Quantity (μg/μL)** |
1 |
11 |
100 |
0.95 |
0.62 |
1.5 |
3.8 |
2 |
11 |
1000 |
5.7-2 |
3.2e-2 |
1.8 |
2.3 |
3 |
5 |
1000 |
4.2e-2 |
2.4e-2 |
1.8 |
1.7 |
4 |
2 |
1000 |
2.7e-2 |
1.6e-2 |
1.7 |
1.1 |
5 |
1.5 |
1000 |
2.4e-2 |
1.9e-2 |
1.3 |
0.97 |
6 |
1.5 |
1000 |
2.8e-2 |
1.8e-2 |
1.5 |
1.1 |
Table 2: Absorbance on remains of total RNA samples, performed on January 20, 2005.
Total RNA
Sample |
Amount of yeast
culture originally
taken (mL) |
Dilution
factor (RNA:
TBE Buffer) |
A-260nm |
A-280nm |
Ratio (260/280)
Using 100ul |
RNA Quantity
(μg/μL)** |
1 |
11 |
100 |
0.666 |
0.35 |
1.90 |
2.66 |
2 |
11 |
100 |
0.554 |
0.274 |
2.02 |
2.22 |
3 |
5 |
100 |
0.748 |
0.367 |
2.04 |
2.99 |
4 |
2 |
100 |
0.369 |
0.178 |
2.07 |
1.48 |
5 |
1.5 |
100 |
0.06 |
0.02 |
3.00 |
0.24 |
5 |
1.5 |
100 |
0.083 |
0.044 |
1.89 |
0.33 |
5 |
1.5 |
100 |
0.15 |
0.73 |
0.21 |
0.60 |
6 |
1.5 |
100 |
0.194 |
0.096 |
2.02 |
0.78 |
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.