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Sunday, June 13, 2010

My Work As a Molecular Biologist


by AP


My workbench where I did primer design, computations,read  literatures and of course analysis.....



Multiplex PCR the one I've missed to use

For confirmation of primer design( checking for size amplicon size and troubleshooting)

My baby:  Roche qRT-PCR  where I did quantitative gene expression analysis 


RNA room: RNA extraction, PCR  stock preparations, cDNA synthesis and normalisation. my dwelling place.haha




The sight outside of the RNA room of our laboratory....

Saturday, May 29, 2010

Fossil Hunt and Naturalist Expedition

Palawan, Philippines- I am in Palawan this week to collect fossils in a nearby Batang batang River, the same river where I used to swim when I was a kid. I will also collect pictures of indigenous plants and animals in Palawan. I will post them in this blog next week.

Friday, May 28, 2010

Human Instinct

Beauty is defined by symmetry. Most of us adhere to that general concept. When looking for a potential mate my female friends prefer a handsome looking guy or an intelligent guy. The same goes with my male friends except that they are more critical  with the physical appearance. It is an animal instinct to look for a more superior trait in a potential partner to ensure reproductive success and be part of the ongoing evolution.





















Phenotype or the physical appearance of an individual is dictated by what is encoded in the DNA sequence. Human being can sense good traits from a potential partner even at the first few seconds of encounter. We can gauge or seize up the person if he or she can be your partner or lover. It is indeed a human instinct.

Summer Professional Researcher

I've worked as a summer researcher of the Tissue Culture and Biotechnology of the International Rice Research Institute(April 15-May 30,2010). I worked again with iron transporter genes in rice. The experiment went successful.

Friday, May 21, 2010

Detailed Steps on Primer Design

Detailed Steps on Primer Design
I list here the steps on how to design a good working primer.
1. Get the sequence of your gene from published literature for example OsNAS3.
2. Using the TIGR Blast/ NCBI Blast, check the whole sequence if it specifies the gene of interest. It is important that the gene sequence is 100% homologous to the plant where the sequence is derived and the most important is that the whole sequence specifies only one distinct gene.
3. Put your sequence inside the box of Primer 3 online software. Specify in the software what are the parameters of the primer you want to have. Click generate. You would get something like this.
Important Notes:
When designing primers take note of the following:
GC content, Tm o primer, 3’ ends of the primer
PCR product size- It depends on what your purpose is. If your going to used the conventional PCR you can create a primer with a product size of 400bp and up. If you’re going to use the primer for real time PCR design a primers with 100-350 size only.
4. To be extra sure if your primer is really specific, Blast them again with NCBI Rice Blast. This would look like this. With the use of Vector NTI/Bioedit you can check the size of your PCR product.
5. You are now ready to order your primers at SBS or proligo.

Thursday, May 20, 2010

Gene Expression Results

I am getting various trends in gene expression of the genes I am studying. Some genes tend to be express early on the development while others were highly expressed on the later stage




Kingston 4 GB Class 4 SDHC Flash Memory Card SD4/4GB

Monday, May 17, 2010

Beach in Southern Palawan



Here is a video of a beach I took from Narra, Palawan. It portrays the rich mangrove forest of Narra facing the South China Sea. On the background includes the fire we set up to cook the fish that we were suppose to catch.

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Sunday, May 16, 2010

Plasmid Standard Preparation by Serial Dilution

Following the method of Applied Biosystems we arrived at this computation but still we were not able to get a good standard instead we got a noisy standard. Please help us solved this problem. Please comment here.

How can we improve our standard?

Copy Number Determination Optimisation Using Real Time PCR Machine

The objectives of this study is to  check what gene is suitable for creation of a standard,to establish real time PCR as a high throughput technique for copy number work and to optimize / prepare a good standard for absolute quantification analysis for this work.
We designed two strategies for the optimization of qRT-PCR. The first one is through Southern blot copy number correlation with qRT-PCR. The second is through the creation of standard using a plasmid carrying the construct that was used in the plant.
In lieu of the first objective to find a gene suitable for doing copy number work we encountered problem with NOS gene. 

We encountered problem like this using plasmid standard. Please look at the illustration.
One proposal to solve  problem is the use of MS2 RNA. We still haven't tried it yet.

We did the serial dilution of the samples following the computation by Applied Biosystems. Click the link for computation.

Enzyme Assay for Glutathione Reductase Improved


Glutathione reductase assay GR activity was determined following the procedures of Halliwell and Foyer (1978) with slight modifications, by measuring the decrease in absorbance at 340 nm. The reaction mixture with a total volume of  1 ml contains 100 µl of    0.5 M  potassium phosphate containing 1 mM EDTA (pH 7.8), 810 µl of distilled water, 50 µl of crude extract, 20 µl of 10 mM NADPH in 50 mM potassium phosphate containing 1 mM EDTA (pH 7.8) and 20 µl of 10 mM oxidized glutathione(GSSG) in 10 mM potassium phosphate. The components were added in the 1-mL quartz cuvet   in the same order with GSSG as the last component. Since the enzyme extract was crude, 2 minutes was allotted for the nonspecific oxidation of NADPH before adding the GSSG. The reaction mixture was carefully mixed and read immediately at 340 nm  at an interval time  of 1.5 seconds for a  total time of  300 seconds using the DU 800 Spectrophotometer All measurements were made in triplicates. The activity of the enzyme was calculated using molar absorptivity constant of NADPH (6.2 mM-1cm-1). The activity of glutathione reductase was expressed in terms of µmol NADPH oxidized per mg protein per minute.


-Blue Lab

Friday, May 7, 2010

What is Acute Renal Failure?

What is acute renal failure?
It is the RAPID  DECLINE in Glomerular Filtration Rate(GFR) over hours and days causing deterioration in renal function  resulting to build-up of nitrogenous wastes. It causes a sudden retention of endogenous and exogenous substances (e.g. Nitrogenous wastes). Reduction in urine flow rate aka oliguria <400ml/day and electrolyte and acid-base abnormalities are its common symptoms/effects.
Classification
Acute renal failure is classified into three namely Prerenal, Intrarenal, and  Postrenal renal failure. Prerenal ARF or azotemia causes renal hypoperfusion, resulting in decreased function without frank parenchymal damage  which accounts for approximately ~55% of ARF cases. Intrinsic ARF directly involve the renal parenchyma ~40% of the cases. Postrenal ARF is associated with urinary tract obstruction~5% of the cases.
 Prerenal Acute renal failure
    It is the most common cause of community-acquired ARF due to history of poor fluid intake, treatment with Non-Steroidal Anti-inflammatory Drug (NSAID), ACE inhibitors/ARBs and  worsening heart failure. Common features include blood volume depletion due to absolute/postural hypotension, low jugular venous pressure and  dry mucus or it can be due to  decreased effective circulatory volume(e.g., heart failure or liver disease) and  decreased cardiac output.
  I. Hypovolemia
    A. Increased extracellular fluid losses: hemorrhage
    B. Gastrointestinal fluid loss: vomiting, diarrhea 
    C. Renal fluid loss: diuretics, osmotic diuresis, hypoadrenalism
    D. Extravascular sequestration: burns, pancreatitis, severe hypoalbuminemia (hypoproteinemia)
    E. Decreased intake: dehydration, altered mental status
   II. Altered renal hemodynamics resulting in hypoperfusion
    A. Low cardiac output state: diseases of the myocardium, valves, and pericardium (including tamponade); pulmonary hypertension or massive pulmonary embolism leading to right and left heart failure; impaired venous return (e.g., abdominal compartment syndrome or positive pressure ventilation)
    B. Systemic vasodilation: sepsis, antihypertensives, afterload reducers, anaphylaxis 
    C. Renal vasoconstriction: hypercalcemia, catecholamines, calcineurin inhibitors, amphotericin B
    D. Impairment of renal autoregulatory responses: cyclooxygenase inhibitors (e.g., nonsteroidal anti-inflammatory drugs), angiotensin-converting enzyme inhibitors, or angiotensin II receptor blockers
    E. Hepatorenal syndrome
Causes of Intrarenal acute renal failure
II. Parenchymal (Intrarenal)
     1. Specific
         a. Glomerulonephritis
         b. Interstitial nephritis
         c. Toxin, dye-induced
       2.Nonspecific
         a. Acute tubular necrosis
         b. Acute cortical  necrosis  
I would like to give particular attention on acute tubular necrosis .It is the death of the tubules inside of our kidney and this could be detrimental to the overall function of our kidney.  What  causes acute tubular necrosis?
  A.      Ischemia: causes are the same as for prerenal ARF, but generally the insult is more severe and/or more prolonged
   B.    Infection, with or without sepsis 
   C.   Toxins:
          1. Exogenous: radiocontrast, calcineurin inhibitors, antibiotics (e.g., aminoglycosides), chemotherapy (e.g., cisplatin), antifungals (e.g., amphotericin B), ethylene glycol
          2. Endogenous: rhabdomyolysis (rapid breakdown of muscles), hemolysis
This illustration tells you how severe beating/ hazing or any intense muscular injury could lead to acute renal failure which could be fatal.

Causes of Postrenal Acute Renal Failure
III. Postrenal  -obstruction to the flow of urine
       1. Calculus in patients with solitary kidney
       2. Bilateral ureteral obstruction
       3. outlet obstruction
       4. Leak, posttraumatic
MANAGEMENT
Prerenal ARF
>Hypotonic solutions (e.g., 0.45% saline) are usually recommended as initial replacement in patients with prerenal ARF due to increased urinary or gastrointestinal fluid losses(Fluid replacement).
>isotonic saline may be more appropriate in severe cases
> Cardiac failure may require aggressive management with ionotropic agents, preload and afterload reducing agents, antiarrhythmic drugs, and mechanical aids such as intraaortic balloon pumps
INTRINSIC ARF
>Many different approaches to attenuate injury or hasten recovery  for ischemic  and nephrotoxic AKI failed.....
>Acute glomerulonephritis or vasculitis may respond to immunosuppressive agents (glucocorticoids, alkylating agents, and/or plasmapheresis, depending on the primary pathology).
POSTRENAL ARF
> requires close collaboration between nephrologist, urologist, and radiologist.
>Obstruction of the urethra or bladder neck is usually managed initially by transurethral or suprapubic placement of a bladder catheter, which provides temporary relief while the obstructing lesion is identified and treated definitively.
>Similarly, ureteric obstruction may be treated initially by percutaneous catheterization of the dilated renal pelvis or ureter.
>Obstructing lesions can often be removed percutaneously (e.g., calculus, sloughed papilla) or bypassed by insertion of a ureteric stent (e.g., carcinoma).
>Most patients experience an appropriate diuresis for several days following relief of obstruction. 

Thursday, May 6, 2010

Science, Nature and Troubleshootings: Troubleshooting and Doing Gene Expression Analysis

http://en.wikipedia.org/wiki/Gene_expression Science, Nature and Troubleshootings: Troubleshooting and Doing Gene Expression Analysis

How To Do Real Time Gene Expression Work for Beginners?

This gene  expression experiment was done using  rice Oryza sativa seeds at various stages of seed  development. The following picture will show you the flow of the work.
Shoot and seed samples of the rice plant were collected and  immediately  frozen in liquid nitrogen. Futhermore, RNA extraction was done using Trizol method with slight modifications like twice the addition of Lithium Chloride precipitation. After that RNA quality and quantity was verified using gel electrophoresis  and nanodrop spectrophotometer. Two intact bands(18s and 28s) of RNA were observed  indicating a good quality RNA. Next the crude RNA samples were treated with Promega RQ1 RNase free DNase.


 For the purpose of clarity, I posted here the picture of the different stages of grain development in rice.
Here are the different tissues we considered for the experiment.

DOWNSTREAM APPLICATIONS
    cDNA synthesis
  Afterwards, cDNA was synthesized   from  the DNase treated RNA using the Roche 1st Transcript cDNA synthesis kit. Here are the components of the reaction: template mixture( oligo dT, random hexamer, DNase treated  RNA) and reverse transcriptase mixture( RT buffer, RNase inhibitor, Deoxynucleotide mix and reverse transcriptase).
Here is the procedure how to do it? First, denature the template mixture for 10min at 65 degrees centigrade and put  immediately in ice for 5mins. Then followed by addition of  the RT mixture to the template mixture. Incubate for 10 min  at  25 degrees centigrade then   followed by 30 min at 65 degrees centigrade.

PRIMER TEST

Primer design and primer check took most of  my time in doing  this work. For the purpose of protecting my Intellectual Property rights, I would not reveal the identity of the genes which I designed and tested. Our research will be published officially this year.

So here is the point in this work.
First, you have to design your primers using bioinformatic softwares. I would discuss in detail how to design primers in my next post. After designing my primers and ordering them, we then test our primers with cDNA using the conventional PCR like G-storm cycler to check PCR product size and the number of band(s) that appeared on the gel. If the primers showed to have one distinct band  and  appropriate product size on the gel, I can now proceed in performing the real time PCR primer check (qRT-PCR). qRT-PCR is highly sensitive and very accurate. In the illustration, after performing melting peak analysis the primers which showed to have two peaks were discarded and  we JUST considered  the primers with ONLY ONE MELTING PEAK . So in short, primers working with conventional PCR will not necessarily work with qRT-PCR. Primer design and specificity is the first priority. 

SELECTION OF REFERENCE GENES

From an original choice of  four reference genes(housekeeping genes), we selected three reference genes to normalize the expression of our target genes based on its expression profile using its Crossing point. The more uniform the crossing point(Ct or Cp) to each other the better it is. Gene stability was checked across the different rice genotypes and its accompanied rice tissues.
We tested the reference genes stability at the different stages of grain development and across different genotypes of rice. We used the geNorm software to evaluate our four reference genes which debunks or removes reference genes with M(relative stability)>1.5 which in this case it was EP1(Ref4). Bioinformatic softwares like geNorm will make the work very easy. Just click on the link.



NOTE:

Contamination should be avoided with all means possible. Always work in  a room designated for RNA extraction only and a room with no possible contamination of plasmids.

 Relative expression computation

Computation of relative expression could be done by means of relative quantification with of course the use of internal reference genes(control genes) and the other method utilizes the power of calculus with is the absolute of quantification method. Pfaffl 2001 suggests the used of an internal calibrator/control sample to normalize the expression of the target sample using the formula of delta delta Ct(relative expression).Relative expression computation can be done in Microsoft excel.  Another method used to compute was through delta Ct method wherein all Ct values of samples or reference genes were converted to raw expression values with used of a control sample or a sample with highest(earliest Ct). These raw expression of the target genes would be normalise by the geomean of AT LEAST three reference genes in order to correct its raw expression (GeNoRM, Vandonsempele, 2000).

Here is the sample gene expression  data we got using relative quantification method  with the use of  at least THREE reference  genes.

Reference:
Pfaffl M.W. , 2001. A new mathematical model for relative quantification in real-time RT–PCR 
Vandosempele et al 2002
 
   
    

Wednesday, May 5, 2010


Troubleshooting RNA Extraction in Seeds Oryza sativa

Extracting RNA from the seeds was never easy as mentioned by one of the PostDocs of the International Rice Research Institute(IRRI). As a researcher of IRRI,  I've worked  with those samples close to three years and indeed STARCH is a big problem in the RNA extraction. Face with this problem, I developed or optimised a protocol on how to extract the RNA with the LEAST amount of STARCH possible. By the way, starch causes one  of  the major problem in downstream applications like conventional PCR and especially real time PCR. One of the procedures, I emphasized is the used of doubly repeated LiCl precipitation. If you want some advice or  if you want a copy of the protocol, please feel free to email me at magponcio@gmail.com.