Hii

After seeing your gel. I can say that may be Degradation or your total RNA prepration for this sample not up to the mark... Please once you just repete this sample.. and see... And this time you try Total RNA, cDNA, and control check in 1 day it wont take long time... avoid to store Total RNA.... And Suppose if you are getting very small pellet in that sample after Isopropanol Step so go for overnight incubation in -20C

Lane 3 of your gel contains less total RNA compared to the other 3 lanes. Even lane 4 has slightly more total RNA compared to lanes 1 & 2. Also lane 3 is not degraded. There is less RNA therefore the 18S band is less visible.

You might have some Trizol present in your lane 3 sample and that would result in a higher OD reading thereby giving you a higher concentration of RNA than you actually have. 

Good Luck!

After quantifying quickly your image  the 28s/18s ratio between samples 2. 3 and 4 it comes out as 2.1 , 5 and 1.41. You do have indeed different ratio, but a contaminant could NOT change that ratio. You don´t mention if all 4 samples are from identical sources, as the 28s/18s is documented to vary between tissues and cells.

Just a reminder that this is only an indirect clue about your mRNA quality! I wouldn´t bother too much about it if the RNA is pure.

Thank you guys so much for your advice :)

@Yugantak Raj Gupta: yes, I did get very small pellets from all 4 samples, so that might be the cause. And is it good to incubate the samples overnight after isopropanol step because degradation would probably occur to some extent?

@Tom Masi: Thank you. And one more point is that sample in lane 3 showed the lowest value among the four samples. Trizol contamination result a higher OD, but could it affect the bands? 

@Petar D. Petrov: I don't know why the ratio is different like this, but as you said, the ratio is not the same. I also used cells from the same source (same passage and culture dish). 

Trizol contamination will not affect your bands/RNA. It would only affect your concentration when you do a 260/280nm OD reading. Trizol is essentially acidic phenol and since phenol is a benzene ring structure it will be read at OD 260/280nm similar to nucleic acids. So if phenol is present in your sample you will get a falsely high reading. It is always a good idea to run a small amount of your sample on a gel to check the integrity and determine if your concentration is accurate based on your OD reading.

Good Luck!

I think that the total amount of rRNA in sample 3 is less. You can do a densitometry sort of estimation by comparing with the known amounts of RNA ladder/standard. As others have pointed out, you perhaps have contamination giving higher OD readings.

Just a recommendation!! I have worked with small RNAs and mRNAs. As a precaution, I never stored them at -20 ºC for more than a week, always at -80 ºC. If I am working with large quantities then I always aliquot them to avoid repeated freeze-thaw cycles.

I agree with Tom and Kshitiz, it seems to be just a question of the amount of RNA you have loaded. And when you have a lower concentration the ratio 260/280 tends to be smaller. Good luck!

Tom Masi, Kshitiz Tyagi, Patricio Fernández-Silva: Thank you so much!

Kshitiz Tyagi: could you share your method for RNA storage? I'm dissolving RNA in RNAse-free water and keeping it at -30oC. How long could your sample can be stored at -80oC? Thank you in advance.

Dan Mai Nguyen,

You should Better store your RNA samples at -80°C in Tris-EDTA buffer (10 mM Tris, 1mM EDTA) and avoid freeze-thaw.

Hi, I think you just have a low RNA concentration in that sample. If you are really worried about the quality you should analyze them on a bioanalyzer (Agilent 2100 bioanalyzer) or Agilent 2200 Tape station, if you have access to either one. This will give you an RNA integrity number (RIN) and is a very reliable way to ensure that you RNA quality is fine before proceeding with downbstream applications. It only uses up 1ul of your RNA or less if you have plenty and can dilute it down. I would not rely heavily on the nanodrop

@Dan, For my analyses it was important to store the RNA in salt-free buffer because I would process them for analysis by LC-MS. I always stored them at -80 ºC in nuclease free water (bought from Life Technologies) in lo-bind nuclease free tubes (bought from Eppendorf). You can of course use DEPC treated water and tubes too.

In my opinion it pays to be extra-paranoid for RNA samples and prevent any contamination. While preparing samples, I would extra clean my bench and cover it with cling-film. Keep a separate set of pipettes, tips, tubes and reagents, away from dust and any contamination.

@Kshitiz Tyagi: Thank you so much for  sharing your tips :)

Hi. I am facing a similar problem for one of my samples which shows an almost invisible 18S RNA. This problem has occurred following DNAseI treatment/AcidPh:CHCL3/EtOH pptn of the initial total RNA sample which had intact 28S,18S and 5S RNA bands on 1% agarose gel but showed slight genomic DNA contamination which made DNaseI treatment necessary.  

However, after purification following DNaseI treatment, I see only  intact 28S and 5S bands and extremely faint 18S band. Unfortunately, I don't have more cells to repeat RNA extraction, so I would like to know if anyone has ever proceeded with cDNA synthesis and RT-PCR with such a sample. If so, how were the results and CT values for endogenous/housekeeping genes and specific target genes? 

@Anurupa Devi:

Hi Anurupa, 

I also used other samples (not the ones I mentioned in this topic) and treated my samples with DNaseI. However, I didn't carry out electrophoresis, but measured OD values instead. Some of my samples had very low OD due to the loss of RNA during extraction process, but I still used them for the cDNA synthesis. I used 5X PrimeScript RT master mix (Takara), and the recommended amount is 500ng of RNA, but I had only ~300ng. The Ct values, then (of course) varied by 2 cycles for both housekeeping genes and target genes.

Hope this info helps.