7 replies to this topic

[wntiong]

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.

[stylothecancer]

wntiong, on Oct 20 2009, 11:20 AM, said:

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?


Perhaps you can try to use vacuum concentrator to concentrate your sample.  

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 think should be no problem; worth a try.  Ya, the calculation is correct.  
What are the special things i need to take caution during microarray? Thanks for the advise.

I am not used to Nimblegen platform; so I can't provide you any specific precaution on the platform.
Generally, the most important thing is the quality of your sample; in this case, your RNA sample quality, which should be intact and pure.  Normally I would recommend people to use the Bioanalyzer to check the RNA sample.  If using bioanalyzer, if your sample is having the RIN between 8 to 10, then you can proceed the your experiment without any query and with confident.  But sometime decision need to be made if it is at the gray zone of RIN 5 to 7.

Besides that, you should do QC to check the quality of your labeling prior to hybridization, which I think Nimblegene would suggest the steps.  Besides that, the temperature of any incubation process would be critical, so check the temperature first prior to begin your experiment.

I hope this would help.  Anyway, would be really appreciate if you could provide me additional detail of the kit or array you are going to use.

[gogreen]

wntiong, on Oct 20 2009, 08:50 AM, said:

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[]  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

[stylothecancer]

gogreen, on Oct 21 2009, 08:46 PM, said:

wntiong, on Oct 20 2009, 08:50 AM, said:

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[]  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!!!"

[wntiong]

stylothecancer, on Oct 22 2009, 05:33 AM, said:

gogreen, on Oct 21 2009, 08:46 PM, said:

wntiong, on Oct 20 2009, 08:50 AM, said:

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[]  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.

[gogreen]

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.
[/quote]

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?

[wntiong]

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

[gogreen]

When I was working on Agilent platform, we were using as low as 100 ng per labeling. But right now I am working on Affymetrix platform and the current protocol used in the lab uses 9 micrograms of poly A RNA!!! That is around 2.5 milligrams of total RNA needed to start the process!!

I don't have any prior experience with Nimblegen, Maybe u should ask the people from the company themselves what would be the lowest amount that you can go! or move to another platform which can do with lesser amounts of RNA. When working on clinical samples,I guess that would be the better way in long run since it would be a PITA to collect these huge amounts of RNA