Real time PCR

While the results of my in situs are very fascinating, it is also important to quantify relative levels of gene expression in embryos that have been injected with my Notch constructs. Thus, another major project that I undertook this summer was getting started with semi-quantitative real time PCR (polymerase chain reaction), also called qPCR. qPCR functions kind of like normal PCR in that a set of primers is used to amplify a sequence of interest from a cDNA library. However, it is different in that a molecule called SYBR green is also in the reaction. During the amplification step, this SYBR green binds to double stranded DNA. Only when it is bound to DNA does it fluoresce, and this fluorescence is measured by a detection device on a machine called an ABI one-step real time PCR machine. Thus, you can use the detected levels of fluorescence to determine the relative amount of amplified cDNA present. How does this all work? The basic steps are as follows:

1. Obtain tissue of interest. In my case, this means injected embryos at the 2-cell stage as I normally would and then freezing them in liquid nitrogen at stages of interest from neural plate stage to swimming tadpole stage.

2. Extract RNA from the tissue. This is a major step of the protocol and will be discussed in further detail later in this post.

3. Use the RNA to make a cDNA library. This involves using a kit with primers of sequences that occur frequently in RNA transcripts. The primers bind to the RNA, and an enzyme extracted from a retrovirus called reverse transcriptase then uses the RNA as a template to make cDNA.

4. Combine a sample of cDNA with primers, SYBR green, and Taq polymerase, and conduct PCR! PCR cycles the reagents in your tube through different temperatures so that your sequence of interest is amplified. The first temperature is extremely hot (92 degrees Celsius) so that two strands of complementary cDNA will denature. The second is a cool temp so that primers can anneal to single strands of cDNA. The final step is warmer (82 degrees C) so that the DNA polymerase isolated from a species of archaea that lives in hot springs (Thermus aquaticus) can bind to the primers and replicate the 100-120bp sequence of interest. This is the portion where SYBR green is detected.

This is an extremely delicate experiment and can only be used to detect expression of genes in a relative manner (what is the difference between samples, or how is one gene expressed relative to another in a given sample of cDNA). Primer design is extremely important as primers are temperature sensitive and must also not anneal to themselves or to each other as this will cause the SYBR green to fluoresce. I spent a considerable amount of time during the spring semester designing primers and was able to run a test real time experiment.
I spent several weeks this summer attempting to optimize a protocol for RNA extraction from my injected embryo tissue samples. RNA extraction is extremely difficult because cells in an organism are equipped with lots of machinery to degrade RNA, since the cell produces much more RNA than it could possibly use. This equipment is mostly in the form of RNAses, which must be quickly inactivated and removed to prevent degradation of RNA samples. RNA is also less biochemically stable than DNA because of the hydroxyl group on the 2′ carbon of the ribose ring. It is also very sensitive to alterations in salt concentration, so buffered solutions are utilized in its extraction. I spent several weeks attempting to use an RNA extraction kit from a company called Qiagen and followed up with a DNAse treatment with a kit made by the company Ambion. Adding a DNAse to the RNA sample is a crucial step. This step eliminates genomic DNA that could get amplified and/or could bind the SYBR green and cause a false signal. After altering various parts of this protocol including alterations to the amount of organic solution used to extract proteins from the sample as well as altering the concentration of DNAse, I landed on a decent protocol. However, RNA is still extremely difficult and very sensitive to degradation. We as a lab have begun to look into using a machine called a MagMax that uses magnetic beads to extract RNA in successive solution washes. Currently, I am in collaboration with our lab tech Zoe and another member in the lab to optimize this protocol. The excessive amounts of yolk present in the younger embryos is also a barrier because it contains lots of lipids that are difficult to extract. Hopefully we’ll find the answer soon!