500-700 ng of total RNA is not too far off from 1 ug. Besides using 1000 ng RNA as starting material for RT is not an absolute requirement. The starting material for RT ranges between 0.1 pg - 5 ug. You can safely proceed with your PCR with 1-2 uL of cDNA obtained from 500-700 ng of RNA.

500-700 ng of total RNA is not too far off from 1 ug. Besides using 1000 ng RNA as starting material for RT is not an absolute requirement. The starting material for RT ranges between 0.1 pg - 5 ug. You can safely proceed with your PCR with 1-2 uL of cDNA obtained from 500-700 ng of RNA.

The mass requirement is dependent on your kit or protocol. Most good kits have a range, like 1 ng to 2.5 ug. I doubt your RT step would be so strict. Under the assumption that it is this strict, use your 4 uL and convert all of it to cDNA. If you have water to add in the protocol, reduce it's volume by 2 uL and your reaction should proceed fine. The usual reaction volume is 20 uL, so you would now have a theoretical 500-700 ng of cDNA in that 20 uL. If your downstream application requires a certain concentration of cDNA, it should be easy to adjust from there. 

Now for option two. It is usually difficult to quantify your cDNA after RT, as there is always RNA and ribonucleotides left over (RNase clean up can help, but it is usually unnecessary), so going two additional cycles may be potentially damaging, or cause other problems. Furthermore, the enzyme in the reaction tube is reverse transcriptase, and cannot (efficiently anyway) transcribe DNA template, only the potentially degraded RNA from the first synthesis round. This is not to say you cannot try, it really depends on the guidelines of your protocol/kit you are using. If you are already doing multiple RT cycles, I see no issue in this, you just may want to add some additional deoxyribonucleotides to be sure of complete cDNA synthesis. 

It also depends on your downstream application; is it RT-qPCR? If that is the case you have more than enough to analyze your target minigene plus up to 4 reference genes (not counting standard curves however). That is usually good enough for normalization.What is your downstream application?

Thanks for all the solutions. To the second answerer, after the reverse transcription step, i will be doing PCR followed by gel electrophoresis to assess test exon inclusion or exclusion.

So i guess its possible then to troubleshoot by doubling the cdna template amount used in PCR. I wonder why my mentor never suggeated this to me. Any further advise is much appreciated!

I dont know if this helps but the reverse transcription kit used is MULV reverse transcription kit. PCR kit im using gotaq polymerase.

Also isit really a case that the RNA to cdna conversion is roughly 1:1? Im using oligo dt primer and ive been thinking multiple primers can bind to multiple sites on poly A tail.

Roughly, as I have been told it is close to 1:1. This is because reverse transcriptase has  some RNase activity. So what you have is a first strand that is synthesized and perhaps some truncated cDNA transcripts. It is important to realize that no matter how well optimized the system is, there are always artefacts and variables that skew things one way or the other. In your case all you need is a positive or negative response on your gel (is there a band or isn't there) and perhaps amplicon size (are you doing sequencing too?). For this your starting template should be a fair amount, but with an optimized PCR you should be able to get good yields with double digit ng (10-50 ng) amounts depending on amplicon length and other variables. Gotaq Hot start polymerase seems to have a quality control standard where it amplifies a starting amount of 0.35 ng of a 2.4 kb gene to produce a 360 bp product. You will want to get a handle on how to optimize your pcr if it has not already been done, but normal protocols might work fine, you won't know for sure until you try. Best of luck!

RM: Also is it really a case that the RNA to cdna conversion is roughly 1:1?

No, and it is because mRNA comprises only 1-3% of total RNA. Therefore reverse transcription of 1 ug of total RNA will not yield 1 ug of cDNA.

RM: I am using oligo dt primer and I have been thinking multiple primers can bind to multiple sites on poly A tail.

Since oligo(dT) primers initiate reverse transcription at the 3´ end of the transcript, difficult secondary structure(s) may lead to incomplete cDNA synthesis, hence yield is not greatly affected by multiple primers binding to multiple polyA tails on mRNA molecules.

Dr. Ali, Great point!! How could I have messed that up! oligodTs will not efficiently prime ribosomal RNA or other non-coding RNA (unless they also have long adenine repeats too). The rRNA is definitely the dominating constituent  which is always clearly visible in agarose gel. What I should have intimated was that the mRNA present generally generates a single copy for so called "first strand synthesis" RT. But as Syed said, this comprises a very small percentage of the overall RNA extracted. 

I have definitely learnt a lot from these exchanges. thanks a lot. Now it gets rather tricky. I mean out of the remaining 400-700 ng of total RNA, there isin't much mRNA , and out of this total mRNA, how much does it constitute my minigene products. 

I may not end up with enough mRNA to begin with. But i will double the cDNA volume used in the PCR and see how it goes on monday. Worse to worse i shall repeat  from the pre-DNAse step sample again. Will let you guys know the outcome.

 It worked. Thanks a million to each and every one of you 

Great. I am glad you got it to work.