10th wk blog

Subject: Cytogenetics

Name of the Tests: Overnight Harvesting of Amniotic Fluid (AF) Sample using BRDU
HI people it’s me, TING-JIE again. This is the 10th week, and we are already half way of our SIP program, hope everyone having fun at their work place.

This week I am going to blog about what we will do after we set up the Amniotic Fluid (AF) sample in cytogenetics laboratory (which I blogged in the last blog entry). The next step after setting up is the harvesting of AF sample.

The purpose of harvest is to obtain sufficient cells at the metaphase stage with chromosomes of acceptable lengths. Remember my last blog I mentioned that for standard cytogenetic procedure, the cells being studied must be at late prophase or metaphase for analysis. That why the harvest step have to carry out to arrest cells in the metaphase.

Method:
1. Add 25 µl of 2.5mg/ml BrdU (Bromodeoxyuridine) and 25 µl of Colcemid® working
solution (1 in 12 dilution) to the culture dish

2. The next morning with a transfer pipet, remove the culture medium completely

3. Gently add 2 ml of warm 0.8% Na citrate hypotonic solution and let it stand at room temperature for 30 minutes

4. Prepare fresh cold 3 parts of methanol and 1 part of glacial acetic acid fixative, mix well

5. Gently add 1 ml of cold fixative to pre-fix the cells and leave for 2 minutes

6. Use a vacuum pump to discard the hypotonic-fix mix , and slowly add 2ml of cold fixative,leave to stand for 20 minutes

7. discard fixative and add another 2ml of cold fixative, leave to stand for 20 minutes

8. repeat the procedure with another 10 minutes cold fixative

9. remove the fix and blow dry with a fan until it is completely dry

10. check the chromosome spread quality with a phase contrast inverted microscope

11. gently remove the cover slip with a pair of splinter forceps

12. mount it with 1 drop of mounting medium onto a pre-labeled microscope slide with the cell side facing upwards

13. bake the slides for 1 hour at 90℃

Now the slides are ready for staining using routine G-banding method, which I will explain in the next blog.

Principle of the test:

Colcemid® is the mitotic arrestant. It prevent the formation of spindle fiber, which normally pull the sister chromatid to opposite poles for incorporation into the 2 daughter cells. Therefore it works by stopping cells in synthesis, and collect a large population of cells ready to head into division together, so that we can get more metaphases.

However, colcemid® will cause the shortening of chromosome or chromosome condensation, when the chromosome condense, the subbands merging into bands, to prevent the shortening of chromosomes a releasing agent has to be added.

Releasing agents such as thymidine, which will produce a greater number of bands as it inhibit chromosome condensation. In our lab, we use BrdU (Bromodeoxyuridine) which is an analog of thymidine as the realeasing agents.

Treatment with the warm 0.8% Na citrate hypotonic solution is to increase cell volume as it swell the cells and enable the cells to disperse wider, thus facilitate chromosome analysis. It works by creating concentration gradient across the cytoplasmic membrane so that the water will rush into the cells by active transport. To increase the effectiveness, pre-warming the hypotonic solution can speed up water transport across the cell membrane and also softening the cytoplasmic membrane.

Pre-fix the cells help to harden the cells and preserve the chromosomes which make cells more resistant to the shock of the pure fixative.

Fixation procedure removes the water from the cells, kills and preserves them, hardening the membranes and chromatin. It also prepares the chromosomes for the banding procedure later.

Yap that’s all for this week, Hope you all understand=)

TingJie
Tg02
0608495h

Week 9

Hi All,

How's everyone doing? Its Week 9 of SIP, and it seems that we are almost halfway through there..just 11 more weeks to go! Fast as it seems to be, its like we just started SIP and I believe all of us are cracking our heads over MP.

So I am currently in Specimen reception department, which is actually a admin dept. So I aint gonna talk about admin stuff. What I will share will be Serology dept, the dept that I was in 2 weeks ago.

Basically, almost all companies now uses automated machines to assist us in our work. Technology is really wonderful in such a way that it does things for us, leaving with much, much lesser things to do. Most of the practical work that we learnt in school are not applied in labs, due to the fact that manual work are no longer utilised in labs.

IMMULITE 1000

Immulite 1000 is used to measure two types of hormones levels in our body - DHEA & HGH. Therefore, it consists of two components:

• Immulite DHEA-SO4


• Immulite IGF-1

Immulite DHEA-SO4 measures dehyroepiandrosterone sulfate (DHEA-SO4), which is an adrenal sulfate in our body.

DHEA-SO4 is, in fact, a sulfated version of Dehydroepiandrosterone (DHEA), a steroid hormone that is thought to be the 'superstar' of all hormones.

Thus, the concentration of DHEA-SO4 relates to the amount of concentration of DHEA, as DHEA-SO4 is 500 times more than DHEA.

You just need the patient's blood serum to do this test.

DHEA:

DHEA is a natural-occurring hormone that decreases with age. It is produced in the adrenal glands and it may be also possible to be derived from the testes. It is said to be the precursor of testosterone and oestrogen, as they share similar chemical properties. DHEA levels increases & peaks when one reaches adulthood, but drop as one starts to age after his 40s. Thus, many diseases which correlate with age also correlate with low levels of DHEA production.

Immulite IGF-1 measures insulin-growth factor (IGF-1) levels, which is stimulated to be produced by Human Growth hormone (HGH) itself.

HGH:

HGH is a hormone that is produced in the pituitary gland & released only when the hypothalamus 'signals' for it. HGH levels decreases as one gets older. It promotes growth in the young by mainly promoting cell growth and height increases during youth. HGH is highly significant in the body as it is thought to be associated with 'youth'. As HGH decreases, the ageing process increases.

Thus, the immulite system involves determining the top two hormones that are involved in ageing. As such, the system is used to predict whether the patient is :

• Suffering from growth disorder
• Taking pills to supplement growth
• Undergoing anti-ageing treatment

Principles:

The immulite 1000 system utilizes assay-specific, antibody, or antigen-coated plastic beads, as the solid phase, alkaline phosphtase-labeled reagent, and a chemiluminescent substrate. The coated bead, is found a proprietary plastic device, namely a test unit by Siemens.

Test unit

Antibody/antigen-coated Plastic bead

The test unit serves as a reaction vessel for the immune reaction, the incubation and even washing. The bead and sample's serum is first placed in a test cup, before being placed in the roller to enter the machine.

Sample Cups

Roller to place the sample cups, before entering the machine

After incubating the bead with the sample's serum and the alkaline phosphatase reagent, the reaction mixture is separate from the bead by spinning the Test unit at high speed on its vertical axis. The entire fluid contents (the sample, excess reagent and wash solution) is transferred to a coaxial waste chamber in the Test unit. The bead is left with no residual, unbound label. The amount of bound label is then quantiated with a dioxetane substrate that produces light. Light emission is measured by a Photomultiplier Tuve(PMT) and the results are calculated.

The results are given by values. There is a list of reference values for all age group by decades. However, there is no significant value for DHEA & HGH. Any value is acceptable, as deficiency or exceeding HGH & DHEA hormones does not cause any disease/harm to the body.

Any qns, feel free to ask me. I know its abit long, and lengthy. But I have no choice.

Lloyd Lam 0607775D

SIP Week 8 Sharing

Subject: Lab Technique

Name of Test: Faecal Extraction

Hi people! Because I am in a research lab, I don’t get a chance of rotating around the different labs. And so, yes, I am still having my attachment at the endocrine lab.

Well, most of the time I am doing my MP. So, in this post, I am going to talk about a lab technique that is frequently being carried out in the lab for my MP.

Faecal Extraction. Yup. You guys didn’t read wrongly. Its faecal extraction. It is not really that smelly.

But before I go into that, let me discuss about my MP first. My MP has got to do with the clouded leopards. Clouded leopards are presently being classified as vulnerable and their actual numbers are unknown as there hasn’t been much research done on them. And because their numbers are declining, there are many conservation projects that are being undertaken by the zoos. However, the breeding process shows little success. This has been thought to be due to stress. Hence, for my MP, my main aim would be to establish their stress level and hope to be able to provide information to help reduce the stress brought about to the leopards.

To help analyse the stress levels, the hormones that we are measuring are glucocorticoids - cortisol and corticosterone and the samples used were faeces.

Why faeces?
This is because it can allow a more accurate result obtained. The collection of faeces for testing is a non-invasive method. Hence, it does not induce stress to the animal which could eventually lead to bias results. Urine could be another sample option but as we all know, it’s kind of hard to collect urine especially from such a fierce animal. Blood samples are not used as this is an invasive method in which it will tend to induce stress to the clouded leopards and thus could result in biasness in the results obtained. Furthermore, it is impractical to collect blood regularly.

Before we can measure the hormones, we need to extract steroids from the faeces first. This is done using the boiling method.

Method:
1. Dry the faeces using a lyophilizer for about 2 days.
2. Pound the faeces into powdered form using a pestle.
3. Weigh out 0.200 ± 0.002g of faeces into the test tubes.
4. Add 4.5 ml of ethanol and 0.5 ml of distilled water to the test tubes.
5. Mark the levels of the solution in the test tubes.
6. Vortex briefly.
7. Boiled the test tubes in the water bath at 95˚C for 20 minutes.
8. Add 100% ethanol as needed to prevent the test tube from boiling dry.
9. Bring the volume up to pre-boiled level using 100% ethanol.
10. Centrifuge the samples at 2500 rpm for 20 minutes.
11. Pour off the supernatant into another set of clean test tubes.
12. Add 4.5 ml of ethanol and 0.5 ml of distilled water to the test tubes.
13. Centrifuge the samples at 2500 rpm for 20 minutes.
14. Pour off the supernatant into the test tube containing the first set of supernatant.
15. Dry down the second set of test tube using a water bath and purified compressed air.
16. Add 1 ml of PBS and vortex briefly.
17. Sonicate for 15 minutes.
18. Pour the extract into 12 x 75 mm plastic tubes and store them at -80˚C.

Hope you guys understand. =) How to go about analysing the hormones level will be explained in the next post.

Enjoy.

Xin Yi
TG02

the 7th week of SIP =)

Hi people! Hope you guys are doing fine for SIP. =)

I'm Lyn and yes, I am back once again after 7 weeks of attachment. I know almost everyone is saying this but still, time really flies.

In my very first post, I had already mentioned briefly on what I do in the lab that I am in. This time round, I shall share with you guys on blasting. As I am in a research lab, I won't be changing departments now and then. Thus, the things that I do are more or less the same.

Blasting
Ok. Blasting is a step that the lab I'm in does after the sequencing step. Sequencing is done by a DNA analyser. After the whole sequencing procedure is done, the trace files for a particular primer is retrived by injecting your thumbdrive into the desktop that is linked to the DNA analyser to save them. Usually, you have to will retrieve 2 trace files in total for a primer. This is because we do sequencing for both forward and reverse.

Trace files are the end result that you get after sequencing is done. By transferring the trace files and opening them in sequencing programmes, you can get the electropheragram of each DNA sequence. In the lab I am in, the sequencing programmes they use are Sequencing Analysis version 5.2 and SeqScape version 2.5 (Applied Biosystems, USA).

As these programmes enables one to view the electropheragram, we are able to bast. Blasting means detecting SNPs in the different DNA sequence. By doing blasting, we are able to find out any known or new SNPs in the gene. Blasting is actually quite an eye-straining step to do. However, there are no alternatives to it. One has to look through all the DNA sequences to detect the SNPs.

How one detect SNP(s) is bascically through scanning the whole DNA sequence of each different DNA. A SNP is shown on the electropheragram as two peaks at the same base nucleotide position. Usually, only one peak is seen.

Sometimes, blasting is made tougher when there are a lot of 'noises' in the DNA sequence. 'Noise' in this case is peaks formed by excess reagents that interfere with sequencing. Usually these 'noises' will affect the jugdement of whether the peak is a SNP. This is because, some of the 'noises' are of such high peaks that one may think or assume that they are SNPs. According to what I'm taught, one can confirm a peak is a SNP when the peak is almost the same height as the peak of the correct base, or at least 2/3 the height of the peak of the correct base.

By comparing with the reference sequence of the gene, one can detect and confirm the polymorphisms in a gene. Reference sequence of a cetain gene most of the time shows the correct DNA sequence that is found in the majority of the healthy population.

When a SNP is found, one will record down or comment on the position of the SNP and also the change in base mucleotides, in the obtained reference sequence of the gene. Reference sequences of different genes can be found at the UCSC genome browser webiste (http://genome.ucsc.edu/).

That's all. I shall share with you guys more on what I am doing in my lab the next time i post again. Hope what i explained was clear.

Take care everyone and have fun! =)

Lyn
0611027D
TG02

Weeks V and VI Attachment

Hello again Y’all!



Indeed six good weeks have flown by and it’s my blogging once again, just right before I knew it. Hope all is fine there and that all your lil’ test tubes are still returning every cheeky grin of yours.

Alright, all things proper, in the previous entry, a brief overview of the major project was posted. So you’d probably know by now what metformin is and some theory about HPLC. With that, in this entry, I‘d be putting up some stuffs involved in the method development stage of the project.

Preparation of an Extemporaneous Metformin Sample

These procedures here are practically the same as most other usual practices in the lab. The only difference is that the sample arrives in a tablet form instead of the usual powder form; thus an additional step is required here, followed by the usual.




Materials:

• 500mg metformin tablet
• DI water
• sonicator
• Mortar and pestle
• 100ml volumetric flask
• glass funnel
• glass stirring rod
• 5ml dropper
• filter paper (optional)***

Methods:




1. Thoroughly pulverize the metformin tablet into powder with the mortar and pestle.*

2. Rinse the drug particles off the pestle into the mortar with DI water.

3. Dissolve the powder using the stirring rod.











4. Transfer the drug solution into the volumetric flask using the glass funnel.

5. Rinse any remaining drug particles from the mortar into the volumetric flask.


6. Top up the rest of the volume with DI water till the 100ml mark, using the dropper when nearing the end.






You might be able to see a faint white line on the bottle immediately to the left.

















A filled volumetric flask.




7. Shake the volumetric flask vigorously to dissolve any remaining powder in the solution.

8. Place the volumetric flask onto a sonicator** to aid in the dissolving process and removal of bubbles in the solution sample.













9. Filter the solution (optional) ***.


* You might notice that there is no weighing of any samples here to be dissolved, unlike that in the preparation for the standard sample. Instead, the entire tablet is used for the making of the sample.

** A sonicator is a machine that causes sonication, whereby sound (ultrasound) energy is used to agitate a sample for various samples such as that of speeding up the dissolving of a solute and the removal of gases (bubbles) from the sample.

*** Filtering is necessary for the sample in this experiment it would be used for HPLC. Thus, it would be removal of any undissolved particles would be necessary to prevent interference with the outcome of the results.

In this experiment, this sample is usually used as an unknown sample, whereby it is used to compare against a calibration plot from the standard and determine if it is accurate. In later phases of the project, it might also be used to facilitate a typical sample that would be made by patients themselves in stability tests.




Preparation of a Metformin Standard Sample

This procedure here uses a commercially available metformin standard, for the preparation of a solution with a concentration of 500ppm*. The procedures as you might probably notice are slightly different from that for the preparation of an extemporaneous metformin sample in that weighing is involved.

Materials:

• Meformin standard

This is the one we have in the lab.

• DI water
• Electronic pan balance
• 50ml** volumetric flask
• watch glass
• glass funnel
• glass stirring rod
• 5ml dropper
• spatula
• filter paper (optional)

Method:


1. Weigh out 25mg of metformin standard on the watch glass using the electronic pan balance.
2. Transfer the sample into the 50ml volumetric flask.
3. Rinse off any remaining metformin standard particles on the watch glass into the volumetric flask with DI water using the glass funnel to help collect the solution.
4. Top up the sample in the volumetric flask to 50ml, using the dropper when nearing the end.
5. Shake the sample vigorously to dissolve to metformin standard.
6. Sonicate the newly formed solution sample.
7. Filter the sample (optional).



* ppm denotes “parts per million”. The actual definition of it is quite complex and thus a simpler way of understanding it in this case is by looking at some examples such as 1mg/ 1000ml = 1ppm, 500mg/1000ml = 500ppm, 250mg/500ml = 500ppm etc. Essentially, the number of milligrams over a thousand milliliters is the number of parts per million.

** A 50ml volumetric flask has been selected here as it provides a sufficient amount for the carrying out of various tests within a suitable period of time while reducing unnecessary wastage of the standard (the standard is very expensive!).

In this experiment, the prepared metformin standard sample is usually used to chart a calibration plot for the determining of any samples of unknown concentrations and also to aid in determining the suitability a particular set of conditions for the quantification of metformin. Its concentration would usually be diluted to lower concentrations. Some commonly used concentrations are 50ppm and 100ppm.


An example of how dilutions are done is in the case of 50ppm for example, whereby 1ml of the 500ppm standard sample is transferred into a 10ml volumetric flask, with the rest of the volume being topped up with the mobile phase or DI water. Thus, simple calculations would confirm that 500ppm/10(DF) = 50ppm.

Alright, that’s all for this entry. Thanks for taking the time to read it and hope you’ve benefited somewhat. All the best to the rest of your attachments once again!

Alexander Soo TG02
0608122H