Difference between revisions of "20.109(S11):RNA to DNA by RT-PCR (Day5)"
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The RT in RT-PCR here stands for reverse transcription, that is, making DNA from an RNA template. We will use a 1-step RT-PCR kit from Qiagen, though the two procedures (reverse transcription and PCR) can be performed separately. The Qiagen kit utilizes a cocktail of two different RT enzymes: Omniscript and Sensiscript, the latter optimized to detect very low abundance sequences. After reverse transcription, these enzymes are inactivated, and a heat-sensitive polymerase is activated so PCR can begin. In sum, the reactions will run for two hours, and we'll spend this time meeting with the writing across the curriculum faculty. | The RT in RT-PCR here stands for reverse transcription, that is, making DNA from an RNA template. We will use a 1-step RT-PCR kit from Qiagen, though the two procedures (reverse transcription and PCR) can be performed separately. The Qiagen kit utilizes a cocktail of two different RT enzymes: Omniscript and Sensiscript, the latter optimized to detect very low abundance sequences. After reverse transcription, these enzymes are inactivated, and a heat-sensitive polymerase is activated so PCR can begin. In sum, the reactions will run for two hours, and we'll spend this time meeting with the writing across the curriculum faculty. | ||
− | One somewhat unusual component in your RT-PCR reactions today is BSA, or bovine serum albumin. In the pilot experiments for this module we found that heme itself (which you used during your column elution last time) inhibits the RT-PCR! The inhibition may be due to hydrophobic interactions between heme and the polymerase; in any case, adding a hydrophobic protein such as BSA that should compete with this interaction seems to work. In Prof. | + | One somewhat unusual component in your RT-PCR reactions today is BSA, or bovine serum albumin. In the pilot experiments for this module we found that heme itself (which you used during your column elution last time) inhibits the RT-PCR! The inhibition may be due to hydrophobic interactions between heme and the polymerase; in any case, adding a hydrophobic protein such as BSA that should compete with this interaction seems to work. In Prof. Niles s original work, he used a different RT-PCR kit that did not face this inhibition issue, which goes to show that sometimes very subtle factors are at play when an experiment goes wrong! Trouble-shooting is a key part of learning to do research. |
[[Image:20109_Protoporphyrin_IX.png|thumb|right|200px|'''Protoporphyrin IX.''' Public domain image from Wikimedia Commons.]] | [[Image:20109_Protoporphyrin_IX.png|thumb|right|200px|'''Protoporphyrin IX.''' Public domain image from Wikimedia Commons.]] | ||
− | + | [[Image:20109-JN_bind-curve.jpg|thumb|left|300px|'''Sample heme binding curves.''' From top to bottom, the curves are 8-12, a mixture of 6-5 and 8-12, 6-5 — each with heme — and heme alone.]] | |
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− | + | One week from now, you'll perform the aptamer-heme binding assay, so now is a good time to take a closer look at heme. Heme and similar compounds contain a large porphyrin or proto-porphyrin ring (see image above right). The functional unit of porphyrin is a pyrrole, an amine heterocycle; four pyrroles make up a porphyrin. As you may know, conjugated aromatic rings often exhibit interesting spectroscopic or fluorescent properties. Heme s natural absorbance maximum, or Soret peak, is at about 396 nm. RNA molecules that do not bind heme do not affect the location of this maximum, though they may slightly shift the height of the entire curve. However, heme-binding aptamers shift the Soret maximum to about 405 nm, and also substantially increase the magnitude of the peak (see image above left). | |
− | Proptoporphyrin IX is the direct precursor to heme: the enzyme ferrochelatase adds a single iron ion to the center of the PPIX ring to make heme. Heme biosynthesis is interesting in its own right, as this system is an example of negative feedback (see original reference [http://www.ncbi.nlm.nih.gov/pubmed/5935350 here (1966)] and further reading [http://www.ncbi.nlm.nih.gov/pubmed/8978272 here (1997)]). In other words, an increased concentration of heme in a cell reduces further heme production. (How do you think such a system might work?) In total, 8 enzymes are required for heme biosynthesis, some acting in the cytoplasm and others in the mitochondria. Defects in heme pathway enzymes result in a series of diseases called | + | Proptoporphyrin IX is the direct precursor to heme: the enzyme ferrochelatase adds a single iron ion to the center of the PPIX ring to make heme. Heme biosynthesis is interesting in its own right, as this system is an example of negative feedback (see original reference [http://www.ncbi.nlm.nih.gov/pubmed/5935350 here (1966)] and further reading [http://www.ncbi.nlm.nih.gov/pubmed/8978272 here (1997)]). In other words, an increased concentration of heme in a cell reduces further heme production. (How do you think such a system might work?) In total, 8 enzymes are required for heme biosynthesis, some acting in the cytoplasm and others in the mitochondria. Defects in heme pathway enzymes result in a series of diseases called |