RNA extraction from tissues

Procedures for RNA extraction from tissues:

1. pre-weigh an empty cryovial
2. take the tissue specimen out from the cryovial and place it on a 100mm petri dish
3. use a scapel and sterican (like fork and knife) to cut the tissue into desired size
4. transfer the tissue into the pre-weigh cryovial and weigh again (this will give the exact weigh of the tissue being process)
5. transfer the surplus tissue into the original cryovial and keep in the nitrogen tank
6. add 650µl of RLT buffer into the cryovial
7. pound the tissue using a pipette tip until the solution turn orange in colour.
8. transfer the solution into QIA shredder column and centrifuge at 14 000rpm for 4minutes
9. transfer the flow through into a new eppendorf tube and add in 550µl of 70% ethanol
10. centrifuge the eppendorf tube at 14 000rpm for 3 minutes
11. transfer the solution into Rneasy column and centrifuge at 14 000rpm for 15 seconds
12. discard the flow through
13. add 700µl RW1 buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
14. discard the flow through
15. add 500µl RPE buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
16. discard the flow through
17. centrifuge the Rneasy column at 14 000rpm for 1 minute
18. transfer the Rneasy column into a new sterilised round bottom tube
19. add 30µl Rnase-free water and centrifuge at 14 000rpm for 1 minute
20. transfer the flow into the column and centrifuge at 14 000rpm for 1 minute
21. the flow through is the RNA extracted

Thing to note
- after taking the cryovial of tissue out from the nitrogen tank, keep it in liquid nitrogen.

Yeo Sok Kian Jocelyn

0703359J

Immunochemistry- G6PD quantification

Hey guys, sorry for the late posting! This will be the last time I'm sharing! (:
This time I am going to talk about the quantitative determination of G6PD on an analyzer, which is currently undergoing evaulation.

Principle
The principle of the assay is to utilize G6PD (that is present in the RBCs) that catalyzes the oxidation of glucose-6-phosphate (G6P) to 6-Phosphogluconolactone (6PG). This takes place in the presence of NADP, which will be reduced to NADPH. The NADPH produced will then react with a colour reagent to produce a fluorescence that will be measured colourimetrically at 550nm.

What is G6PD
G6PD is an important cytoplasmic enzyme that is found in cells and plays a crucial role in reducing the oxidative effect of the free radicals on the RBCs. It is involve in the first step of the hexose-monophosphate pathway (HMP) to produced NADPH, which is essential in maintaining the integrity of the RBCs membrane. Hence, with a deficiency of this enzyme, the RBCs are prone to lysis due to the oxidative stress. And this may lead to hemolytic anaemia.

Why do this test?
An awareness of G6PD deficiency had been raised many years back in Singapore. Hence, all newborns will be screened for this deficiency. And of course, the samples used for this test will be all cord blood samples.

How does the assay work?
First, all the samples will be transferred manually into eppendorf tubes and loaded onto the analyzer. Next, an elution buffer will then be added to the blood to lyse the RBCs. This is to bring out the enzyme, if it is present. The reagent solution is then added for the production of NADPH in the presence of G6PD. After which, the colour reagent will be added for colour production if NADPH is produced. Incubation will take place before the absorbance reading is taken.

The quantitative results will then be calculated after the readings are entered onto Excel. For deficient samples, they will be re-run again to confirm whether if the enzyme is really absent.

That should be all! (: Feel free to ask me any questions, I will try my best to answer them.

Posted by: Tan Siew Ming
0702862D

research: BCA assay

it's week 16! how's everyone doing!

this week, i'm going to explain what is BCA protein assay.

what is BCA protein assay? it is an assay that quantitates protein concentration.

what does BCA stands for? BCA stands for bicinchorinic acid.

what is the principle of this assay?

principle of this assay:

2 major reactions occur in this assay

1. proteins are held together by peptide bonds. upon exposure to copper ions (Cu2+), the peptide bonds will reduce Cu2+ to Cu+. this reduction process is temperature dependent. this also means that, the more proteins you have in the solution, the more copper ions are reduced to Cu+.

2. BCA molecules will bind to Cu+, resulting in the formation of a purple-coloured product. the colour intensity of this product is then measured at wavelength 562nm using a plate reader. (colourimetic assay)

how does the assay work?

the assay usually comes in a kit. inside the kit, you can find: BCA reagents A and B, 96-well flat bottom plate, Bovine . Albumin (BSA) standards.

BSA standards: as the name suggests, BSA is a serum albumin, and we all know that albumin is a protein. so the BSA standards serve as a control for the kit (standard control). the BSA concentration that the company provides is usually 2mg/ml (2000ug/ul). to get a series of concentrations, we dilute the BSA (from high concentration to low).

Range of concentration of BSA diluted: from 0mg/ml to 2mg/ml. usually we can have up to 6-7 standards inclusive of 0mg/ml. BSA is diluted in ultrapure water (Milli-Q water).

Eg,

well A: 0 ug/ml (no BSA standard) /BLANK

well B: 131.072 ug/ml BSA

well C: 262.144 ug/ml BSA

well D: 327.68 ug/ml BSA

well E: 409.6 ug/ml BSA

well F:512 ug/ml BSA

well G: 640 ug/ml BSA

well H: 2000 ug/ml BSA

next, we prepare the BCA working reagents .

BCA working reagents = Reagent A + Reagent B (in ratio 50:!)

reagent A is colourless, reagent B is blue in colour.

one important thing to note is that the working reagent must be prepared freshly prior to use and stored at 4 degrees celsius. Do not keep overnight.

upon adding reagent B, the solution immediately turns GREEN. mix well.

the next component we will require is THE BUFFER. this buffer, simply refers to the the buffer we use in our samples. eg, if the samples are cells, then the buffer is the cell culture medium.

steps:

1. Add 20 ul of BSA standards to the individual wells.

2. Add x ul of sample to each well.

3. Add x ul of buffer to the wells that contain BSA standards.

4. Add 20 ul of Milli-water to the wells that contain samples.

Steps 3 and 4: This is to eliminate disturbances caused by any possible absorption by the buffer when measuring the absorbance of samples. Similar reason applies to adding 20uL of water into the samples’ wells.

5. Add (200-20-x) ul of BCA working reagents to all the wells.

- therefore total volume in each well: 200 ul.

-upon adding, you will find that the solution turns purple in the presence of proteins

6. Mix well, by pipetting up and down.

7. Cover the plate.

8. Incubate at 37 degrees celsius for 30 minutes.

- before reading on the plate reader, ensure that there are no bubbles. the presence of bubbles will affect the reading.

9. Read at wavelength 562 nm.

Simple? Yeah.

Any alternatives?

some of you may have used before the bradford assay. For those who have not, bradford assay is an alternative to BCA protein assay. (Read here for more information on bradford assay (wiki) : http://en.wikipedia.org/wiki/Bradford_protein_assay )

BCA protein assay is better than bradford as:

1. more sensitive compared to bradford assay

that's all! (:

LIM JIA HUI (JOEY)

0703605f tg01

group 2

CK-MB test

Creatine kinase (CK) is a dimeric enzyme which occurs in four different fours: a mitochondrial isoenzyme and CK-MM( muscle type), CK-BB (brain type) and CKMB.

The determination of CKMB is an important element in the diagnosis of myocardiac ischemia. CKMB is detectable in the blood about 3-8 hours after the onset of cardiac symptoms and can be detectable over a period of time which is dependant on the course of the condition.

Test principle

Sandwich principle

1. 1st incubation: 15uL of sample, a biotinlylated monoclonal anti CK-MB antibody, and a monoclonal CK-MB specific antibody labeled with a ruthenium complex react to form a sandwich complex.
2. 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin
3. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then washed away.
4. Results are determined bia a calibration curve which is instrument-specifically generated by 2 point calibration and a master curve provided via the reagent barcode.

Note: The entire test is done using the analyzer (Cobas e) and the test principle can be found inside the CK-MB kit.

Stanley