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Dear all,
I’m trying to quantitate changes in some microRNAs in Zebrafish using the Ncode kit (Invitrogen, for RT) followed by SYBR-qPCR and I need an internal control. Most papers use U6, but the primer sequence is not mentioned, or worse, the primers seem not to amplify U6 but to other genes. For example, in this work, http://www.sciencedi...041008X12003493 , in table 1, the primer set
U6
For
60
TCGCTACGGTGGCACATA
Rev
TATGGAGCGCTTCACGG
actually targets to the gene >NM_201213.1 Danio rerio glutaminyl-tRNA synthetase (qars)
I don't think zebrafish U6 and qars are the same gene, or am I getting something wrong?
Do you know any primers for zebrafish U6 that works in SYBR-qPCR?
Or other choices to serve as an internal control for normalization?
Thank you in advance.
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[Question] primer sequence for zebrafish U6 in SYBR-qPCR for miR normalization?
19 October 2012 - 01:33 AM
[Question]DNaseI in treating neonatal rat cardiomyocytes; size of the cells?
04 September 2012 - 08:24 PM
Hi,
I have some questions about handling neonatal rat cardiomyocytes and I’d appreciated your input.
For those who have cultured neonatal rat cardiomyocytes, do you use DNase I before filtering the cell suspension after collagenase treatment? What’s the amount of DNaseI you used and for how long?
In addition, for people who have counted the cell surface area of these cardiomyocytes, in what size it usually is (um^2)?
The values counted from our lab members sometimes vary a lot, so I just think that data from a third-party might help us in this point.
Thank you very much.
I have some questions about handling neonatal rat cardiomyocytes and I’d appreciated your input.
For those who have cultured neonatal rat cardiomyocytes, do you use DNase I before filtering the cell suspension after collagenase treatment? What’s the amount of DNaseI you used and for how long?
In addition, for people who have counted the cell surface area of these cardiomyocytes, in what size it usually is (um^2)?
The values counted from our lab members sometimes vary a lot, so I just think that data from a third-party might help us in this point.
Thank you very much.
[Question] how to tell FVB from ICR?
03 September 2012 - 10:54 PM
Hi everyone,
We got a problem in our lab now. A batch of FVB mice was put together with ICR mice, and they look the same in appearance.
Does anyone have any method to tell ICR from FVB mice?
Is Fv1b gene uniquely expressed in FVB but not ICR? Or is there any other marker gene or SNP? Or do the two strains behave differently?
This colony is important to us. Please help. Thank you in advance.
We got a problem in our lab now. A batch of FVB mice was put together with ICR mice, and they look the same in appearance.
Does anyone have any method to tell ICR from FVB mice?
Is Fv1b gene uniquely expressed in FVB but not ICR? Or is there any other marker gene or SNP? Or do the two strains behave differently?
This colony is important to us. Please help. Thank you in advance.
[Question] How to remove cell debris stuck on the attached cells in primary card
09 August 2012 - 05:28 AM
Dear all,
I encounter a problem of clung cells/debris while doing rat cardiomyocyte culture. After 2 days of seeding, using DMEM, I tap the dish hard to wash away tissue/cell debris. However, even after more than 5 times of wash, I can still find some cell clumps are very sticky and difficult to remove. Clinging on the beating cardiomyocytes and piling up high, these clumps cause a great trouble for me to take clear images. A picture is attached for your reference.
Is there any way to remove these masses and leave the seeded and attached cardiomyocytes not affected? Thank you very much.
I encounter a problem of clung cells/debris while doing rat cardiomyocyte culture. After 2 days of seeding, using DMEM, I tap the dish hard to wash away tissue/cell debris. However, even after more than 5 times of wash, I can still find some cell clumps are very sticky and difficult to remove. Clinging on the beating cardiomyocytes and piling up high, these clumps cause a great trouble for me to take clear images. A picture is attached for your reference.
Is there any way to remove these masses and leave the seeded and attached cardiomyocytes not affected? Thank you very much.
[Question] how to make primary-cultured cells evenly distributed?
04 August 2012 - 11:13 PM
Dear all,
I got a problem in making primary-culture cells well distributed when seeding!
I’m doing rat cardiomyocytes culture.
The tissues has been subjected to collagenase for half an hour of digestion, and filtered by a cell strainer (100um) before seeding. But I get cells very close to each other, or tangled. (I intended to attach a photo(133Kb) to show you what it looks like, but the system said I'm not permitted to upload this kind of file and I don't know why.)
I want them to be single cells with clear border for I’ll have to count the cell surface area, but the cells distributed as patches, making it hard to outline each cell.
Any ideas to improve the condition? Thank you very much in advance.
I got a problem in making primary-culture cells well distributed when seeding!
I’m doing rat cardiomyocytes culture.
The tissues has been subjected to collagenase for half an hour of digestion, and filtered by a cell strainer (100um) before seeding. But I get cells very close to each other, or tangled. (I intended to attach a photo(133Kb) to show you what it looks like, but the system said I'm not permitted to upload this kind of file and I don't know why.)
I want them to be single cells with clear border for I’ll have to count the cell surface area, but the cells distributed as patches, making it hard to outline each cell.
Any ideas to improve the condition? Thank you very much in advance.
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