You extracted RNA from a spider leg and you quantified your RNA extraction. You

Question

You extracted RNA from a spider leg and you quantified your RNA extraction. You got the values of:

32.5 ng/ul

1.9 = 260/280 ratio

0.2 = 260/230 ratio

Is this a “good” extraction? Why or why not? (2 pts)

Your partner did another extraction of the same spider leg and he got the values of:

20. ng/ul

1.8 = 260/280 ratio

1.8 = 260/230 ratio

Which extraction would you use for setting up a qRT-PCR (you or your partner’s)? Explain why you chose that extraction. (2 pts)

2. Explain why we used a housekeeping gene in our tomato experiment?

_What is the function of the enzyme, reverse transcriptase, and why did we use it in our RT-qPCR?

Explanation / Answer

The ratio 260/ 280 is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. Ratios of 1.8 and 2.0 for DNA and RNA respectively, are "rules of thumb". The actual ratio will depend on the composition of the nucleic acid.

The 260/230 values for “pure” nucleic acid are secondary measure of nucleic acid purity and are often higher than the respective 260/280 values. Expected 260/230 values are in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm.

In your extranction 260/280 ratio is 1.9 which is fine but 260/230 ratio is 0.2 which is very low, therefore it is not a good extraction. Since your partners 260/280 ratio is 1.8 and also 260/230 ratio is 1.8 which determines more purity, we would choose the partners extraction for setting up qRT-PCR reaction.

House keeping genes are used as internal controls to analyze the relative expression of different genes for gene expression analysis. They are supposed to act as the baseline of gene expression in the sample. The are also an indicator of perfect nucleic acid extraction, quality of samples, quality of PCR.

A reverse transcriptase (RT) is used to generate complementary DNA (cDNA) from an RNA template.The cDNA can then be amplified by polymerase chain reaction.

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