# how to convert ug to uL isolate of RNA? - (Jul/15/2008 )

Hi all,
I have isolate RNA with High pure RNA isolation Kit from Roche. Principally, this method use column to separate RNA from DNA and contaminant. In the last step, column add by 100uL elution buffer to get the pure RNA.
The first question: Is that mean the dilution factor 100?
I continue with spectrophotometer, and get absorbance 260:0,484, the ratio 260/280:1,863, ratio 260/230:1,965. After that , I continue to make first strand cDNA with fermentas kit. The first procedure I must mix : 0,1-5ug total RNA, 1uL oligo dT primer, and DEPC treated water to 11uL. I have question:
How many volume of RNA should I mix?
I hope, anyone help me to answer my question so that I can continue the step..
Ragards,
Fiat 29

-fiat29-

QUOTE (fiat29 @ Jul 15 2008, 05:18 PM)
Hi all,
I have isolate RNA with High pure RNA isolation Kit from Roche. Principally, this method use column to separate RNA from DNA and contaminant. In the last step, column add by 100uL elution buffer to get the pure RNA.
The first question: Is that mean the dilution factor 100?
I continue with spectrophotometer, and get absorbance 260:0,484, the ratio 260/280:1,863, ratio 260/230:1,965. After that , I continue to make first strand cDNA with fermentas kit. The first procedure I must mix : 0,1-5ug total RNA, 1uL oligo dT primer, and DEPC treated water to 11uL. I have question:
How many volume of RNA should I mix?
I hope, anyone help me to answer my question so that I can continue the step..
Ragards,
Fiat 29

The first question: Is that mean the dilution factor 100?
No, it dosen't mean that the dilution factor is 100.

OD260=0.484, it means the concentration of the RNA in the spectrophotometer is 0.484 * 0.04 ug/ul = 0.01936 ug/ul.
It looks like that you directly used part of the 100 ul eluent for UV spectro determination (As you have not mention that you have diluted your RNA). So, 0.1/0.01936 ul ~= 5.17 ul, you need at least 5.17 ul of your RNA for RT.

-zhongmindai-

QUOTE (zhongmindai @ Jul 16 2008, 01:22 PM)
QUOTE (fiat29 @ Jul 15 2008, 05:18 PM)
Hi all,
I have isolate RNA with High pure RNA isolation Kit from Roche. Principally, this method use column to separate RNA from DNA and contaminant. In the last step, column add by 100uL elution buffer to get the pure RNA.
The first question: Is that mean the dilution factor 100?
I continue with spectrophotometer, and get absorbance 260:0,484, the ratio 260/280:1,863, ratio 260/230:1,965. After that , I continue to make first strand cDNA with fermentas kit. The first procedure I must mix : 0,1-5ug total RNA, 1uL oligo dT primer, and DEPC treated water to 11uL. I have question:
How many volume of RNA should I mix?
I hope, anyone help me to answer my question so that I can continue the step..
Ragards,
Fiat 29

The first question: Is that mean the dilution factor 100?
No, it dosen't mean that the dilution factor is 100.

OD260=0.484, it means the concentration of the RNA in the spectrophotometer is 0.484 * 0.04 ug/ul = 0.01936 ug/ul.
It looks like that you directly used part of the 100 ul eluent for UV spectro determination (As you have not mention that you have diluted your RNA). So, 0.1/0.01936 ul ~= 5.17 ul, you need at least 5.17 ul of your RNA for RT.

You are right, I used part of the 100uL isolate RNA directly without dilute it before spectro determination. In the other sample, I get OD260=0,056 without dilute, that's mean concentration is 0.056*0,04 ug/L = 0,00224 ug/uL, If I use the lowest concentration RNA in example 0.1ug, its mean 0,1/0,00224 = 44,6uL. So, I need at least 44.6uL to add mixture,whereas the protocol suggest 0,1ug-5ug RNA. What should I do? Can I continue the next step?
regards,
Fiat

-fiat29-

QUOTE (fiat29 @ Jul 16 2008, 05:59 PM)
QUOTE (zhongmindai @ Jul 16 2008, 01:22 PM)
QUOTE (fiat29 @ Jul 15 2008, 05:18 PM)
Hi all,
I have isolate RNA with High pure RNA isolation Kit from Roche. Principally, this method use column to separate RNA from DNA and contaminant. In the last step, column add by 100uL elution buffer to get the pure RNA.
The first question: Is that mean the dilution factor 100?
I continue with spectrophotometer, and get absorbance 260:0,484, the ratio 260/280:1,863, ratio 260/230:1,965. After that , I continue to make first strand cDNA with fermentas kit. The first procedure I must mix : 0,1-5ug total RNA, 1uL oligo dT primer, and DEPC treated water to 11uL. I have question:
How many volume of RNA should I mix?
I hope, anyone help me to answer my question so that I can continue the step..
Ragards,
Fiat 29

The first question: Is that mean the dilution factor 100?
No, it dosen't mean that the dilution factor is 100.

OD260=0.484, it means the concentration of the RNA in the spectrophotometer is 0.484 * 0.04 ug/ul = 0.01936 ug/ul.
It looks like that you directly used part of the 100 ul eluent for UV spectro determination (As you have not mention that you have diluted your RNA). So, 0.1/0.01936 ul ~= 5.17 ul, you need at least 5.17 ul of your RNA for RT.

You are right, I used part of the 100uL isolate RNA directly without dilute it before spectro determination. In the other sample, I get OD260=0,056 without dilute, that's mean concentration is 0.056*0,04 ug/L = 0,00224 ug/uL, If I use the lowest concentration RNA in example 0.1ug, its mean 0,1/0,00224 = 44,6uL. So, I need at least 44.6uL to add mixture,whereas the protocol suggest 0,1ug-5ug RNA. What should I do? Can I continue the next step?
regards,
Fiat

sorry, I mean the protocol suggest mixture not more than 11uL, whereas I must add 44,6uL to the mixture. so, can I continue next step? How should I do?

-fiat29-