q-RT-PCR Procedure

Download
Share  Add to  Flag Embed
Views:
 
Category: Entertainment
     
 
License:   All Rights Reserved

Presentation Description

Quantitative real time PCR in six easy steps explained. This is the presentation for a Graduate Seminar Course, the presentation last aprox. 5 minutes

Comments

 

By: dr.eshanidewan (10 month(s) ago)

Dear Sir, I would like to have Quantitative RT-PCR presentation. Can you please send me the presentation to my mail ID eshanidewan@yahoo.com. I will be thankful to you. regards. eshani

 

By: JaneRada (17 month(s) ago)

thanks alot for the effort to collect such imprtant informations

 

By: abdallahellabban (19 month(s) ago)

Great well done

 

By: muralichalla (21 month(s) ago)

Dear Sir, I would like to have Quantitative RT-PCR presentation. Can you please send me the presentation to my mail ID mmchalla@gmail.com. I will be thankful to you. Murali Mohan

 
By: masoomehamoozadeh (6 month(s) ago)

Dear Sir, I would like to have Quantitative RT-PCR presentation. Can you please send me the presentation to my mail ID masoomeh.amoozadeh@yahoo.com. I will be thankful to you.amoozadeh

 

Presentation Transcript

Quantitative RT-PCR : 

Quantitative RT-PCR Diana Kiser Introduction QRT-PCR steps QRT-PCR an example Conclusion

Introduction : 

Introduction Q RT-PCR (quantitative Real Time Polymerase Chain Reaction) or Reverse Transcriptase RT-PCR. Used for: Amplify DNA Detect pathogens and mutations Gene expression analysis

STEPS : 

STEPS Extract RNA Set up a PCR Mix (Addition fluorescent dye or probe) http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009. Diana Kiser

STEPS 3. mRNA is copied to cDNA : 

STEPS 3. mRNA is copied to cDNA http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

STEPS : 

STEPS 4. Set the plate 5. Perform PCR in a suitable real-time PCR Machine http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

PCR STEPS : 

PCR STEPS cDNA is denatured +/- 94°C (separate strands) Primers are annealed cooling sample to 50-60°C http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

STEPS PCR : 

STEPS PCR Taq Polymerase extends DNA from the primers; raising temperature to 72°C (Melting phase) Repeat several times. http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

STEPS: 6. QUANTITATION OF mRNA LEVELS USING REAL TIME PCR : 

STEPS: 6. QUANTITATION OF mRNA LEVELS USING REAL TIME PCR http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

Slide 9: 

Diana Kiser 2009

STEPS: 6. QUANTITATION OF mRNA LEVELS USING REAL TIME PCR : 

STEPS: 6. QUANTITATION OF mRNA LEVELS USING REAL TIME PCR http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009. http://www.biosci.ohio-state.edu/~pmgf/documents/biorad_iq_manual.pdf

Slide 11: 

http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

Steps 6. Quantitation : 

Steps 6. Quantitation Fold Change in the target gene= Average Number Experiment/Average Number Control Fold Change in the reference gene= Average Number Reference Gene/ Average Number Control Ratio Target Gene Expression (E/C)= Fold Change Target gene/ Average Number Reference gene http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

PCR an Example : 

PCR an Example Peroxisome Proliferator-Activated Receptor γ (PPARγ) Agonist Rosiglitazone (Rosi) Increases the expression of Very Low Density Lipopreotein Receptor (VLDLR) Gene in Adipocytes.Takeshi Takazawa, Toshimasa Yamauchi, Atsushi Tsuchida, Makoto Takata, Yusuke Hada, Masato Iwabu, Miki Okada-Iwabu, Kohjiro Ueki, and Takashi Kadowaki

1. Extract RNA from Obese mice : 

1. Extract RNA from Obese mice ob/ob mice nsatng.files.wordpress.com/2009/02/obese_mice.jpg

Slide 15: 

2. Set up PCR mix. 3. mRNA is copied to cDNA 4. Set the plate with solutions of genes from obese mice fed with fatty diet and with genes from mice with fatty diet plus Rosiglitazone (drug effective in diabetic treatment) For genes VLDR, CD36, aP2. T. Takazawa et. al. 2009

5. Perform PCR : 

5. Perform PCR ABI prism 7900 http://www.amebioscience.com/7900.htm Retrieved Nov /09 from AME Bioscience site.

6. Quantitation- Results : 

6. Quantitation- Results Fig. 1. Effects of Rosi treatment for 4 days on gene expression of molecules involved in lipid metabolism in WAT from ob/ob mice. ** P<0.01 T. Takazawa et. al. 2009

Conclusion : 

Conclusion q RT-PCR is a method effective for amplification and quantification of cDNA from mRNA and is performed in 6 steps: 1. Extraction RNA 2. Setting up PCR mix (adding Fluorescent Protein) 3. mRNA is copied 4. Setting up the plates 5. Perform PCR ( Denatured, Annealed, Extension) 6. Quantitation http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009.

References : 

References NSATNG. (2009, February 19). Obesity: America’s Growing Epidemic. Retrieved November, 2009, from Nutritional Science’s Website: http://nsatng.wordpress.com/2009/02/19/obesity-americas-growing-epidemic/ R&D Systems, Inc. (2009, March). Proteome Profiler™ Array . Retrieved from http://www.rndsystems.com/pdf/ary013.pdf on November, 2009. AME Bioscience. (November. 2009). ABI Prism 7900ht RT-PCR system. Retrieved from http://www.amebioscience.com/7900.htm Takeshi Takazawa, Toshimasa Yamauchi, Atsushi Tsuchida, Makoto Takata, Yusuke Hada, Masato Iwabu, Miki Okada-Iwabu, Kohjiro Ueki, and Takashi Kadowaki . August 25, 2009. Peroxisome Proliferator-Activated Receptor γ (PPARγ) Agonist Rosiglitazone Increases the expression of Very Low Density Lipopreotein Receptor (VLDLR) Gene in Adipocytes. The journal of biological chemistry, M109.047993. Retrieved from: www.jbc.org on September 30, 2009. Hunt, Margaret. Microbiology and Immunology on-line. University of South Carolina, School of Medicine. Oct. 29, 2009. http://pathmicro.med.sc.edu/pcr/realtime-home.htm Retrieved Nov. 2009. Bio-rad I-cycler manual Website: http://www.biosci.ohio-state.edu/~pmgf/documents/biorad_iq_manual.pdf