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wntiong's Content

There have been 43 items by wntiong (Search limited from 20-July 18)



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#81506 source of sample and qpcr

Posted by wntiong on 28 July 2010 - 12:25 AM in PCR, RT-PCR and Real-Time PCR

hi i have 4 genes to study, and used the normal breast tissue (as positive control) total rna to construct standard curve and to check primer efficiency. i want to use delta-delta ct method. however, only 3 out of 4 genes performed well within the range, while the other seem have very low expression in the postivie control samples. If in this case, should i study that low-expressed gene using other source of sample (other than the normal breast tissue)? will it cause any problem when come to discussion of relationship of these four genes in same samples?

thanks



#77417 how to save sybr green reagents?

Posted by wntiong on 28 June 2010 - 09:36 PM in PCR, RT-PCR and Real-Time PCR

hi,

i'm having short of funding and resource problems. i only have 500 sybr green reactions left and i need to screen for 4 genes of interest and 1 housekeeping gene for 30 samples. Still waiting for my primers to arrive, but previous batch of primers i tested doesn't compatible each other, that means i can't use delta delta ct method. Problems now are i don't have much reactions left, and if i run duplicate and use standard curve method everytime, for about 15 samples i'll need 700 reactions. This is not included the failure of experiment if there is NTC amplification or for primer validation test. Tell me what should i do. One way to save the reaction i thought will be not to run all my experiments in duplicate.
Need your all advise. Thanks!


Which reaction volume are you using? and which qRT-PCR instrument? A simple way I can think of is halving your reaction volume, I've used as little as 10Ál in the past. Of course this depends on your instrument you are using, and you'll have to be very very careful and precise with your pippetting to avoid introducing extra errors.


@almostadoctor,

Thanks! I'm using rotorgene 6000 machine and rotorgene qpcr sybr green mastermix, the minimal volume the kit required is 25ul.



#77276 how to save sybr green reagents?

Posted by wntiong on 27 June 2010 - 09:55 PM in PCR, RT-PCR and Real-Time PCR

hi,

i'm having short of funding and resource problems. i only have 500 sybr green reactions left and i need to screen for 4 genes of interest and 1 housekeeping gene for 30 samples. Still waiting for my primers to arrive, but previous batch of primers i tested doesn't compatible each other, that means i can't use delta delta ct method. Problems now are i don't have much reactions left, and if i run duplicate and use standard curve method everytime, for about 15 samples i'll need 700 reactions. This is not included the failure of experiment if there is NTC amplification or for primer validation test. Tell me what should i do. One way to save the reaction i thought will be not to run all my experiments in duplicate.
Need your all advise. Thanks!



#68620 ELISA doubts

Posted by wntiong on 28 April 2010 - 10:24 PM in ELISA and Immunoassay

Hi i need help on ELISA about:

1. I going to use tissue lysate for ELISA, but if i previously use evan blue to stain my tissue, will this affect the ELISA reading?

2. Have u heard about NGF ELISA kit? What brand would you recommend?

3. Most kits i found recommend to use tissue/cell line cultures for NGF detection, because the plasma and sera contain IgG. How does this affect the ELISA performance?

Need your help.

Thanks.



#68404 Measurement of RNA concentration after Dnases treatment

Posted by wntiong on 27 April 2010 - 08:41 PM in Molecular Biology

DNases don't digest the DNA completely into bases, so there are fragments left over that are too small to amplify/worry about. However, these fragments will still be picked up by the spec.


do you is it ok if i just proceed to reverse transcription without uv spec it? the reading is so funny after i uv spec, it gives me concentration shoot up to 3-5 ug rna, and i dun hv the nanodrop in my faculty. can i just assume i take out 10ul equivalent to 1ug rna and proceed to rt? i prepared 20ul dnased mixture with initial sample 2ug rna. thanks



#67873 High Ct value - what to do next?

Posted by wntiong on 22 April 2010 - 09:56 PM in PCR, RT-PCR and Real-Time PCR

Hi,

I need to validate my primer set so recently i constructed a standard curve (1:5 dilution) from 40ng to 0.064ng/ul cDNA template, and the primer efficiency is about 109%. Problem is i have high Ct value, even from 40ng/ul the Ct is about 30, which is very high. The literature suggested to use higher template amount but i also read that the the amount of template used in qpcr should not exceed 50ng/ul. What could be the problem? Should i design new primer or should i try smaller dilution?

Thanks.



#67867 Measurement of RNA concentration after Dnases treatment

Posted by wntiong on 22 April 2010 - 08:33 PM in Molecular Biology

Hi, i did dnases treatment to all my rna samples before go to rt-pcr steps. I only stop dnases reaction after the treatment at 65c, 10 min and no further clean up was done. Just want to know, should i quantitate my rna after dnases treatment, or i can just directly use the rna for rt without measurement? I used 2 ug total rna and dnased-treated them. Can i just assume after dnases treatment, the amount of rna is the same as what i put for starting?

thanks.



#61573 Expired chemicals - 0.5M EDTA, can be used again after autoclaved?

Posted by wntiong on 07 March 2010 - 06:29 PM in Molecular Biology

Hi, i need 0.5M EDTA for the microarray work, but ran out of time and money to get one 100ml bottle from market. Found one expired bottle in the lab, expired on 2006 but the color still clear (no yellowish). Just want to know can i use it again? even if i autoclave? what is the risk/what problem it may bring up to affect the microarray result? thanks



#60889 no or poor amplification

Posted by wntiong on 02 March 2010 - 03:51 AM in PCR, RT-PCR and Real-Time PCR

Hi all,

I used same cdna template.. but when i optimize my primer using conventional pcr method, i did annealing temp adjustment and mgcl optimization. the problem is sometime i got pretty nice bands, but sometimes i got poor amplification or even no amplification, even i used same primer stock, same mgcl2 and everything is the same. it's really odd how could this occur. any advise? thanks



#55784 No PCR product at all

Posted by wntiong on 18 January 2010 - 08:13 PM in PCR, RT-PCR and Real-Time PCR

try to bring a previously tested positive sample and run it, let's see how it'll go..
this will tell u the source of your problem,
whether its in ur reaction or in ur sample..

if the previousely tested sample gave none, i would go and check my thermal cycler temperature,
if it REALLY measures 95C and 72C and the rest ... ( since u have tried the other options )
this is done with the help of the manufacturer, they will provide you with controls ( s.th like thermometers ) put in the wells of the PCR to check for the temperature...

in our lab we've encountered faint bands and after troubleshooting and excluding all the rest possible factors the problem was with the cycler itself, one raw in the PCR was malfunctional...

can u kindly tell, what is your sample ??


best of luck ;)


thanks. my samples were total rna isolated from whole blood, been dnases treated.



#55662 No PCR product at all

Posted by wntiong on 18 January 2010 - 01:34 AM in PCR, RT-PCR and Real-Time PCR

the reason maybe in ur sample ( inhibitors ) or in ur PCR reaction..
have u run a positve control/previousely tested sample along with urs ?


nope i don't have the positive control. this was my first time run pcr after 2 years. but i did second run this morning, still no result at all, only some smears at the bottom which i think is not the pcr product. my second run i increased the template, primer concentration, no. of cycles and make sure everything added in sequence and correctly. still there is no result. i tried two different primers of two genes, still there is no result. need your advise how to troubleshoot.



#55336 No PCR product at all

Posted by wntiong on 15 January 2010 - 02:48 AM in PCR, RT-PCR and Real-Time PCR

i did 25ul pcr rxn against housekeeping gene, then loaded 10ul of pcr product into 3% gel. The pcr product is designed about 200bp. but, there is no pcr product at all after gel run. what could be the reason? thanks.



#52219 is it necessary to do DNases treatment?

Posted by wntiong on 20 December 2009 - 08:36 PM in PCR, RT-PCR and Real-Time PCR

If you have exon-exon spanning primers then there's no need to do DNase treatment. 10ug? per uL or?

Chris


1ug/ul. need 10 ug in 10 ul for microarray. due to sample scarcity, hard to get this amount for my samples.



#51375 is it necessary to do DNases treatment?

Posted by wntiong on 14 December 2009 - 11:08 PM in PCR, RT-PCR and Real-Time PCR

i got low amount of rna for all my samples (less than 10ug), but got to save them for real time pcr and microarray. i heard it is important to do dnases treatment of sample before real time/conventional pcr to avoid genomic contamination, but after dnases treatment, the sample should be clean up which i think my rna will be lost some in this step. I design my primers span exon-exon junction, will that help to avoid genomic dna amplification if i don;t dnases my sample? thanks



#47421 primer-blast from ncbi

Posted by wntiong on 25 November 2009 - 12:47 AM in PCR, RT-PCR and Real-Time PCR

Thanks guys,

i used both primer3 and primer-blast to design primer for same gene, then blast both forward and reverse primer sequences. Apparently two different set of primers were produced and after blast, the primer3's primers showed e value of 0.0015 to that gene's genomic sequence and about e value = 248 for the next genes. for primer-blast's primers showed same e value of 0.0015, but if against to next other gene, it is about 3.5 of e value.

i am not sure. but since the primer-blast has the option to find the exon-exon boundary, which is better according to above blast results?

i am really a beginner onto this, kindly advise though if the question sounds stupid. lol.

thanks



#47341 primer-blast from ncbi

Posted by wntiong on 24 November 2009 - 05:25 PM in PCR, RT-PCR and Real-Time PCR

Has anyone use this software before?

I am new in pcr primer design and want to design primers for rt-pcr. This software has involve chossing of exon-exon junction for users so i thinking to use it. But, how does it compared with primer3? which is better?

thanks



#44124 Nimblegen microarray - do and don'ts

Posted by wntiong on 12 November 2009 - 12:09 AM in Macroarray and Microarray

I've worked with almost every possible cells and tissues from mammals and a lot of plants and bacteria, But havent done too many of the blood RNA isolations and it was always a hard with the blood..Eventhough there are quite good kits available in the market, the yields that you get are low in most cases. If you are working on clinical samples, 10 ug starting material can be a bottleneck! Why don't you try doing it with lower amounts of RNA. It should still work. If u don't mind, can you pls give me an outline of the nimblegen labeling protocol? What kind of labeling is that, what is the dye used and that kinda stuff?
[/quote]


Yes, 10 ug of starting material is like my 1 patient sample whole total rna gone for microarray. =(
I will use one color labeling protocol



#42330 Glycogen and speedvac drying

Posted by wntiong on 03 November 2009 - 04:07 PM in Molecular Biology

Hi, i need to get 10ul of total rna for my experiment, but very often i tried the speedvac drying method, the rna will loss from 30 to 50%. Since my samples are precious, do you think i can add glycogen into my sample and speedvac together? If can, how much glycogen i should add? Will the glycogen affect the microarray experiment or rna degradation?

Kindly advice. Thanks


Hi wntiong,

if you want to reduce the volume to the required amount, using a speedvac is the best way to go..I don't think you would lose any RNA in that process...But make sure you don't dry it too much and then try to resuspend it, it will be hard to redissolve the RNA once its quite dried up.

Glycogen is a co-precipitant which can be used during precipitations when u have low amounts of DNA or RNA so that you can visualize a pellet of glycogen and its assumed that RNA/DNA will be precipitated at the same place and you alleviate the risk of pippeting that out! I believe using glycogen in a speedvac is not going to help. Glycogen will not affect microarray experiments or cause/avoid RNA degradation.


Thanks,

But the problem is i did lost some of my rna during speedvac without glycogen. how long i should set to make sure no rna is losing? is it because i speedvac too long?



#42231 Glycogen and speedvac drying

Posted by wntiong on 03 November 2009 - 01:02 AM in Molecular Biology

Hi, i need to get 10ul of total rna for my experiment, but very often i tried the speedvac drying method, the rna will loss from 30 to 50%. Since my samples are precious, do you think i can add glycogen into my sample and speedvac together? If can, how much glycogen i should add? Will the glycogen affect the microarray experiment or rna degradation?

Kindly advice. Thanks



#40658 Nimblegen microarray - do and don'ts

Posted by wntiong on 21 October 2009 - 07:25 PM in Macroarray and Microarray

Hi All, I am new in microarray and going to use Nimblegen system to run differential display gene expression profilling next month. However, i need some guide, to make sure a beginner like me can run the experiment as smooth as possible. =)

The protocol requires me to obtain 10 ul of total rna of 1ug/ul, which i have difficulties to obtain this amount now. I only have approx. 12 ug in 20 ul, how to reduce my volume sample into 10 ul?

you can use vacuum concentrator to do this or use a Qiagen minElute column to concentrate the RNA to the desired volume

The protocol includes a step of rnases A clean up, and it strictly requires promega RNAses A (4mg/ml). Due to financial constraint, i think i will just use Fermentas RNAses A (10mg/ml) instead to buy one. We will use 1 ul of 4mg/ml RNAses A to clean up, if i use 10mg/ml RNAses A, is it i just add 0.25 ul?

I guess for 4 mg from 10 mg/ul stock, u should be adding 0.4 ul, not 0.25 ul!!

What are the special things i need to take caution during microarray? Thanks for the advise.


Microarrays are not so tricky as they sound, if you follow the protocol fairly well, you stand a good chance to have a good hyb and data. Nimblegen arrays are printed on glass slides,so u can break it if u drop it[:P] I'd say to practice the hybs on the hyb chambers before u set up the real ones and be check the recommended volumes for the hybs and be sure to avoid any leakage in the hybs otherwise u would end up looking at a bad hyb ...thr are quite a few things to be looked at...as i said first, follow the protocol, u should be fine. I was working with Agilent arrays which is quite similar to nimblegen in terms of processes. Another the most important factor is the integrity of the RNA that yopu use for the experiment. We used to take RIN 7-10 RNA for the hybs, as stylothecancer said 5-7 is a borderline case...btw the RIN depends on what organism are u working too!!

all the best



To gogreen,

totally agree with you...... especially "on RIN depends on what organism are u working too!!!"


Thanks.

I am using total rna from whole blood, and i found difficulties to get good amount from the whole blood, even i have used 5 ml of whole blood, yet average it also give me 12 ug of total rna after spin column. You see, the nimblegen requires me to have at least 10 ug of total rna to proceed, how i going to save some samples for real time pcr validation? That's tricky right now, especially accessible to patient samples not easy in my protocol.



#40379 Nimblegen microarray - do and don'ts

Posted by wntiong on 19 October 2009 - 07:20 PM in Macroarray and Microarray

Hi All, I am new in microarray and going to use Nimblegen system to run differential display gene expression profilling next month. However, i need some guide, to make sure a beginner like me can run the experiment as smooth as possible. =)

The protocol requires me to obtain 10 ul of total rna of 1ug/ul, which i have difficulties to obtain this amount now. I only have approx. 12 ug in 20 ul, how to reduce my volume sample into 10 ul?

The protocol includes a step of rnases A clean up, and it strictly requires promega RNAses A (4mg/ml). Due to financial constraint, i think i will just use Fermentas RNAses A (10mg/ml) instead to buy one. We will use 1 ul of 4mg/ml RNAses A to clean up, if i use 10mg/ml RNAses A, is it i just add 0.25 ul?

What are the special things i need to take caution during microarray? Thanks for the advise.



#39928 concentrating RNA samples

Posted by wntiong on 14 October 2009 - 07:22 PM in General Lab Techniques

suggestions above are all good. just to add, we sometimes use amicon ultra centrifugal filter devices. (Millipore)


I have the same question - my microarray work requires only 10 ul of rna to proceed but i have about 30 ul of rna (after spin column), can i use vacuum concentrator to reduce the volume to 10 ul? I dare not to try ethanol precipitation because everytime i tried, many rna gone.



#38213 rna clean up - what is the rna recovery

Posted by wntiong on 29 September 2009 - 11:02 PM in Molecular Biology

Hi guys, i am isolating rna from whole blood using trizol method, and some of my samples have quite good A260/280 and A260/230 ratio of btw 1.7 to 2.0. In fact, i think it is necessary for me to do a rna clean up using spin column method (Qiaamp rna blood kit), but i am not sure what is the rna recovery % since i only managed to get about 10 ug of rna after trizol extraction, i afraid it will get much lesser after clean up step. FYI, i need at least 10ug for microarray work. Kindly advise. Thanks


Microarray work is much more sensitive to contaminants in your RNA sample than qRT-PCR. You should do a spin column cleanup. If your recovery is too low, consider pooling like samples.


I did the spin column, the band is much clearer now, is just that the amount of rna is half of the amount i obtained after trizol. How to pool my samples during spin column cleanup?



#38195 alternative to microarray

Posted by wntiong on 29 September 2009 - 08:30 PM in Research Idea, Design and Collaboration

My project target is to select potential gene after differential display microarray analysis from whole blood samples. I tried to isolate rna from whole blood using tri-reagent method but often not giving me good quality of rna. Many of the rna are degraded which i think maybe the stabilization protocol isn't work. I spent 8 months to optimize my rna extraction protocol in order to get a intact rna for microarray work, but still can't get a single pure rna. I start to fear that maybe i need to think of other alternative instead of continue troubleshoot my problem (and i don't know where went wrong). I would like to know what alternatives i can do in order to finish my masters. My grant money will be finished soon, and i purchased two set of microarray slide and some elisa kits. Currently i have 5 kits of puregene DNA isolation kit in my lab. Kindly advise. Thanks



#38084 rna degradation - what goes wrong

Posted by wntiong on 29 September 2009 - 12:48 AM in Molecular Biology

after running in 1% gel, my samples all appeared degradation (long smear with no 28 and 18s bands at all). I wonder what goes wrong since i quite confident with my skill and the protocol after few months of trying.

I keep my whole blood sample in tri-reagent in -80, and only isolate rna after one or two months (the max - because lab is too far away from the collection centre). I transferred my samples with ice bag and drove a journey of approx 30 mins, and isolate immediately. Then follow every single step as Tri-reagent protocol required, except that after isopropanol precipitation, i transferred and pool the pellet from aliquots (of same sample) into one tube then only do ethanol washing. This helped me to improve my rna yield. I air dried my pellet and if got remaining ethanol i use pipettor to remove it. Then stored the rna with rnaase-free water in -80c.




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