Dear Sir,

Thank you for your kind suggestion. I have one more doubt. I used fresh rhizome tissue from actively growing plants of 6 months age in field condition (total maturity period of the variety is 7 and half months) and processed immediately in liquid nitrogen and used kit according to the manufacturer's protocol with RLC buffer today. I saw bands in FA gel and also processed the sample for cDNA synthesis using Oligo dT primer. I got the resolution as attached. Does it mean that i can't proceed for gene expression study with this result? Is there any difference between gene expression in green house grown plant of 6 months age and that in field condition of 6 months age? Moreover, we just need 100mg sample and that portion of active growth is still present in the rhizome in tertiary portions even after 7 months if we take sample from field condition before complete senescence, so, what might be the problem with taking sample from such younger bud region of rhizomes? (secondary images were taken with mobile camera)

Some times presence of crocumin dinders the band formation .I don't have the experience with Isolation with plant mini kit. Please give a good internet scarch. You may get your answer. 

Thanks maa'm.

Thank you all for your kind suggestions and relevant knowledge sharing. I will trend any further difficulties if I face any in near future.

I performed RNA isolation using RNeasy Plant Mini Kit today using six samples of turmeric rhizome. I found the resolution of RNA bands as in the attached picture (secondary picture was taken using mobile camera). Can I move on for further cDNA synthesis and RT-PCR?

Judged from your gel picture, I think that the RNA is good enough for you to move on with your RT-PCR experiment.

If you want to check further, you can check out the RIN (RNA Integrity Number) of the RNA samples if you have a proper equipment (ex. Agilent 2100 Bioanalyzer System). The higher the number, the better the RNA's quality (in terms of integrity). See attachment for RIN.

@ Ali,

Do you have more info about this statement: " Those ribosomal RNA (revealed as bands on gels) have been protected inside the ribosomes from endogenous RNase activity."? It is interesting. I am wondering that is mRNA also protected once they associated ribosomes (rRNA/proteins)? I am also thinking that how fast the mRNA produced (from inside the nucleus) will associated with or bind to rRNA/protein after they are transported out of nucleus.

Thanks for your suggestions and the document. @Yuan

Regarding your question "Is there any difference between gene expression in greenhouse grown plant of 6 months age and that in field condition of 6 months age?"

1. You can use plant from either environment for your gene expression experiment. However, it won't be a surprise if you see some difference of global gene expressions between these two group of plants. Because the greenhouse is a controlled environment, while plants grown in open field can subject to stressors (such as color temperature at night). During stress, some environmental stress-related genes can be triggered to express.

2. You need to be aware when you do RT-PCR: some genes only express at certain plant developmental stage.  For example, if a gene only express at month #3, and if you isolate total RNA at month #6, there won't be mRNA of this specific gene for you to do RT-PCR. No RT-PCR result will come out (no band on a gel). To find out when a specific gene is expressed, Northern analysis on plant tissues from different developmental stages should be conducted.

Dear Professor Yuan-Yeu, Thank you for your valuable suggestion. 

I checked the quality of RNA today using nanodrop and used RNase free water as blank which I used for dilution of RNA in the last step as per the protocol. I used 1.5 microliter sample from each for nanodrop measurement. I got the results as attached. I want to know whether to go for cDNA synthesis and if yes then what should be the quantity of RNA template to be used? The RNA samples were from two turmeric variety: G1, G2, G3 are of GNT variety and N1, N2, N3 are of NDH variety.

Hi Dipendra,

"The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA" (see attached Nanodrop instruction, yellow highlight).

I checked the 260/280 ratios from your RNA samples on the sheet you attached. They look pretty good except a couple of samples with less optimum value. Your sample 'L' (you have measured several times) looks wield. Probably you need to re-isolate it.

As for the amount of total-RNA you should use for cDNA synthesis: if you are using a kit, usually the instruction sheet will suggest you a number to use. If not, I don't have a number right out of my head now. You can find it online, and I will get back to you later too.

Have a good weekend.

Sir, Thank you for your valuable responses. I am getting lot more help from you. I have one more question; If I used double the volume of reaction mixture for cDNA synthesis (40 microliter instead of 20 microliter) with each component increased proportionately, is there any need for increasing the time length for cDNA synthesis cycle or it will be enough with same cycle length as for 20 microliter reaction mixture?

No, no need. You can also prepare 2 tubes of 20 ul each, then merge them together later.