Analyzing DNA Expression In Mouse Brain After Arsenic Exposure In Drinking Water
Arsenic exposure in drinking water continues to draw attention to whether or not it is associated with type II diabetes and cancers of the skin, bladder, prostate, lungs, kidneys, and liver. Arsenic has been found to occur naturally in groundwater (toxic) in Taiwan and China and yet type II diabetes also tends to be higher in these areas . Copper and smelting industries tend to have higher levels of arsenic, so it is important to study and find out whether this toxic mineral is causing diabetes and regulating gene expression.
In a previous study conducted at The University Of Texas At El Paso, a total of twenty-four (twelve male and females) mice were exposed to this toxin. They were subdivided into three groups with each containing four males and four females (see table 1). These three groups contained a low arsenic group (5ppm/wk), a high arsenic group (25-150ppm/wk), and a control group. showed that blood glucose levels were not affected by arsenic intake. However, gene expression was affected differentially by the arsenic exposure. Most of the genes affected were those dealing with gene regulation, energy metabolism, psychological regulatory mechanisms, and defense mechanisms. The low dose group showed that seventy-five genes were differentially expressed, generally by up-regulation and to a larger degree. The high dose group showed that forty-three genes were differentially expressed, generally by down regulation and to a smaller degree.
The genes that showed the greatest change in down and up-regulation was used to confirm the results of the previous micro-array .05 level of confidence. Pyruvate Kinase was down regulated by 350%. This gene is a key enzyme in glycolysis and has three isozymes. Isozyme I is found in the erythrocytes and liver. Isozyme II is found in the kidneys. Isozyme III is found in the liver, kidneys, leukocytes, skeletal muscle, and cardiac muscle. ERP29(stress inducible endoplasmic reticulum protein) was down regulated by 397.2%. This gene is activated in response to ER stress, such as, misfolded proteins. Also, it has protein binding capabilities. C-CAM4 (corrective adhesion molecule) was up regulated by 785%. It is believed that the loss of C-CAM can play a role in cancer development. 92% on this genes sequence is identical to C-CAMI and C-CAMII. It is believed that it plays a role in regulating the activity on the C-CAM family and it is a secreted protein.
Method and Materials
RNA Extraction (Ambion RNAAqueous RNA isolation kit)
RNA was extracted from cerebellar tissues previously collected from the control and arsenic exposed mice, and preserved in RNALater. A spectrophotometer was used to find out how concentrated our RNA sample was and how much it is contaminated with protein.
Formula: Concentration of nucleic acids(ug/ml) = A260X 40 X dilution factor (total volume/aliquot volume). For convenience the reciprocal of nucleic concentration (X 1000) is often calculated to determine the volume(ul) that needs to be taken to provide 1 ug of RNA .
Samples containing 2-5 ug of RNA were added to a loading dye containing formamide and formaldehyde and heated at 67°C for 10 min. Ethidium bromide ( 1ul of 500 ug/ml) was added and the sample solution was loaded onto a 1.8% denaturing agarose minigel. Electrophoresis was carried out at 80 volts for 45 min. The gel was then visualized under ultraviolet light, to determine the integrity of the RNA sample. The presence of two compact bands with little or no smearing was taken to indicated the absence of RNA degradation.
Reverse Transcription (Ambion RETROscript Kit)
1-2 ug of total RNA was diluted in 10 ul of sterile water. The poly-T primer oligo-dT(.5ug/1ul) in was added into our samples and heated at 70°C for 10 min. This allows the primers and RNA strand to unwind into a single strand and are ready to be annealed when put on ice for 2 min. 5x first strand buffer(4ul), DNTP(2ul), 100mM DTT(2ul), and Powerscript Reverse Transcriptase were combined together and incubated at 42°C for 80 min. This allows the reaction to be carried out and for the CDNA to make many copies. The reaction is stopped by denaturing the transcriptase at 70°C for 15 min.
PCR - Amplifying cDNA
Run PCR to amplify cDNA segments with primers for specific genes: Pyruvate Kinase, ERP29, and C-CAM4. These genes showed a larger degree of up or down regulation in the previous experiment. A mastermix containing 10X buffer(4ul), 10X Taq(.2ul), 10X DNTP(4ul), and sterile water (28.8ul) for one reaction was created. Mix each sample with cDna (1ul) and reverse and forward primers (1ul of each).
The following primers for the indicated genes were used in PCR reactions:
88F: 5’-cct ctc acc act tgc cca a-3’
368R: 5’ – gtt agt gtg tag gct ccc-3’ =>280bp
285F – 5’ cag cgg cag aga gac att a – 3’
368R – 5’ gtt agt gtg tag gct ccc – 3’ =>83BP
36F: 5’ - cccactgctgtccgttct - 3’
747R - 5’ – ggc gg tgag gat gtt gag - 3’ =>711BP
464F – 5’ ctg gat gtc tgc ctg cg – 3’
585R – 5’ gtc tgt ctc ctt cac acc tg – 3’ =>121 BP
F553: 5’ – ctt ctt cca gca gca gca ac - 3’
1553R: 5’ – ccc aca cgg agg aaa cca c - 3’ => 1000BP
1429F – 5’ ctg tcc cga gga gtc tt – 3’
1582R – 5’ gcc aac ctg tca cca caa caa tc – 3’ =>153BP
Incubate mastermix/primer mixture for 2 min at 95°C to initiate PCR. Carry out PCR for forty cycles of: 30 sec at 95°C(DNA strands separate), 30 sec at 60°C (primers bind to template DNA strand), and 60sec at 72°C(DNA extension).
To determine the amount of gene expression for a specific gene we planned to conduct real-time PCR. The faster the reaction hits the threshold, the greater the gene expression would be as shown in figure 1. We can use this to compare our three groups to one another and have an idea of which group is being up and down regulated.
RNA was successfully extracted from the cerebellum of female (figure 2) and male (figure 3) mice exposed to different concentration of arsenic (see table 1) in their drinking water.
Control – 4.09 ul per 1 ug
High – 6.94 ul per 1 ug
Low – 14.60 ul per 1 ug
Control – 21.55 ul per 1 ug
High – 12.79 ul per 1 ug
Low - 6.76 ul per 1 ug
Two complete PCR reactions were carried out which lead to successful gene products (figures 5 and 6); however the expected gene size was generally not observed. The expected gene sizes are located on figure 5 and above and below figure 6. In figure 5 it can be seen that expected gene sizes were observed in sample C8, E4, and P1. In the second experiment, genes products of the expected size were seen in samples 1L and 2L in the female group.
Our PCR segments were accurate to a certain degree because they were smeared and not of the expected size. Some possible explanations that the expected base pairs were not clearly seen in visible bands is that: 1) The melting temperatures for the primer pairs were not sufficiently high to prevent non specific priming. 2) The primer pairs themselves were hybridizing to one another. 3) One or more of the enzymes being used in reverse transcription or PCR preparation was no longer effective. 4) The cDNA was highly denatured and therefore unsuitable for amplification. 5) Insufficient high quality RNA was extracted, resulting in the production of insufficient template cDNA.
The abundance of low molecular weight amplification products is most consistent with non specific priming effects. Such effects can be eliminated through optimization of PCR conditions and possible by redesign of primers. Thus, although these experiments were not successful as originally envisioned, the results suggests that with greater time and effort appropriate conditions can be found for successful completion of this project.?