Sir really thx for the video, can i leave several questions? 1. if you don't mind, may i ask the name of that handy homogenizing machine? 2. if you are goin to extract RNA from many samples, than how you prevent cross-contamination during homogenizing? you wash that homogenizer probe by 70% ethol? 3. is that machine safe enough not to breaking 1.5ml e-tube? i'm using general eppendorf 1.5ml tube now with stock the samples in their
Glad to be of service. Yes, you must wash between samples OR use disposable tips. The Eppendorf tube will not crack as the blade is inside the ferrule. I do not want to promote any specific brand, but here is an example. www.omni-inc.com/omni-micro-homogenizer.html Stay Biosafe during Covid19. Thank you
Hi Kenneth, I am thinking of switching over to using a homogenizer instead of liquid nitrogen and mortar and pestle. Do you happen to know which brand homogenizer you're using?
Please check the following link: homogenizers.net/collections/rotor-stator-homogenizers?view=all Do not use a homogenizer when processing pathogenic biological agents. They generate a significant volume of aerosols. I use one with a disposable rotor to avoid cross contamination.
thank you sir for your video it helped a lot. i used same procedure and kit to isolate RNA from plant leaves, but unfortunately it is contaminated by gDNA and also concentration is very low. what need to avoid this problem. and is there any need to add betamarcaptoethanol to extract RNA from leaves of oak tree?
Thank you Sir. The answer depends on what you intend to do with the RNA during the next step. For cDNA synthesis and RT-qPCR cleanup with DNase is definitely required prior to first strand synthesis. I noticed that if the initial sample size is large, the DNA gets carried over. For tree leaves, try to use the young leaves as the fully mature leaves are loaded with phenolic compounds which inhibit most enzymes. All the best!
Thank you Ms. Carla. Advisable to perform DNase treatment after final elution. The DNase must be RNase free. You may notice a drop in RNA yield. All the best.
I am extracting RNA from fish scales. I took one scale per sample. But the RNA yield was too low and it's homogenization was also difficult. Please help. What can I do to get results?
The RNA concentration will be very low in developed scales. You may have to modify the experimental design. Depends on what your objectives are. Quick suggestion: try flash freezing in liquid Nitrogen.
This depends on the nature of the endosperm. If liquid as in the case of coconut, you will have to reduce the volume to obtain higher yield using a non destructive method as far as RNA is concerned. If solid, there should be no problem. Most endosperm tissue is triploid (3n), this must be considered when analyzing RNA seq data from endorsperm transcriptomes.
@@KENNETHFRANCISRODRIGUES thanks a lot doc. Actually I need to do RNA extraction of maize at 5, 10 and 15 DAP, so I think on 5th and 10th day, it might be milky. What other changes I can make. Thanks a lot for reply.
Thank you for your query. The procedure described above is suitable for the extraction of RNA for downstream application to reverse transcription and reverse transcription quantitative PCR (RT-qPCR), however, it is recommended that a sterile fume hood and the use of appropriate RNase inactivating agents (DEPC) be resorted to in case the RNA will be subjected to RNA sequencing (RNAseq) or in the case of gene transcripts which may be present at a very low copy number in the biological sample.
Thank you for your query. You will have to use Proteinase K (RNase free) during the lysis step. Commercial kits include this in their RNA extraction procedure.
It can use for RNA clean-up purpose. After Phase-separation using chloroform, take off the aqueous phase and mix with ethanol, subsequent step is same the this method.
When I follow the provided protocol and this video, RNA was always contaminated by gDNA . I did all steps on the ice, I changed kit, centrifuge machines and all solutions (EtOH). But still have gDNA on RAN tube. How can I solve this problem....?? And I don't want to treat DNase because of the yield decrease
Dear viewer. Thank you for your comments. Generally, you need to perform a DNA removal procedure using DNase. One of the reasons for DNA carryover is overloading with the initial sample. Begin with a small sample volume. Generally for animal tissue less than 200 microlitre volume of tissue.
![[Biol 515] Module 1 - Part A: RNA Extraction from E. coli](http://i.ytimg.com/vi/sIR3YVPK6IQ/mqdefault.jpg)
Thank you Sir. It is very clear and anyone new in research can follow your step :)
Thank you.
Thank you! It's my first time using this kit and your video helped me a lot.
Lyna Duante Glad to be of service. All the best.
Sir really thx for the video, can i leave several questions?
1. if you don't mind, may i ask the name of that handy homogenizing machine?
2. if you are goin to extract RNA from many samples, than how you prevent cross-contamination during homogenizing? you wash that homogenizer probe by 70% ethol?
3. is that machine safe enough not to breaking 1.5ml e-tube? i'm using general eppendorf 1.5ml tube now with stock the samples in their
Glad to be of service.
Yes, you must wash between samples OR use disposable tips.
The Eppendorf tube will not crack as the blade is inside the ferrule.
I do not want to promote any specific brand, but here is an example.
www.omni-inc.com/omni-micro-homogenizer.html
Stay Biosafe during Covid19.
Thank you
KENNETH FRANCIS RODRIGUES - i do really appreciate your kind comment
Stay healthy, hope you sir got beautiful research works
Thank you
Dear Sir, thanks a lot. do you any video related to cDNA synthesis for qPCR
Hi Kenneth, I am thinking of switching over to using a homogenizer instead of liquid nitrogen and mortar and pestle. Do you happen to know which brand homogenizer you're using?
Please check the following link: homogenizers.net/collections/rotor-stator-homogenizers?view=all
Do not use a homogenizer when processing pathogenic biological agents. They generate a significant volume of aerosols.
I use one with a disposable rotor to avoid cross contamination.
@@KENNETHFRANCISRODRIGUES Can I use homogenizer for extraction from pineapples leaves to extract virus RNA
thank you sir for your video it helped a lot. i used same procedure and kit to isolate RNA from plant leaves, but unfortunately it is contaminated by gDNA and also concentration is very low. what need to avoid this problem.
and is there any need to add betamarcaptoethanol to extract RNA from leaves of oak tree?
Thank you Sir. The answer depends on what you intend to do with the RNA during the next step. For cDNA synthesis and RT-qPCR cleanup with DNase is definitely required prior to first strand synthesis. I noticed that if the initial sample size is large, the DNA gets carried over. For tree leaves, try to use the young leaves as the fully mature leaves are loaded with phenolic compounds which inhibit most enzymes. All the best!
If we do not have an electric homogenizer, what else we can use to homogenise the samples?
Thank you, Sir!
Please, can I add one step for DNA digestion by using Buffer RDD and DNase I?
Thank you Ms. Carla. Advisable to perform DNase treatment after final elution. The DNase must be RNase free. You may notice a drop in RNA yield. All the best.
Thank you!
What concentration of RNA we can expect by this method ?
Approximately 50 - 200 nanograms when measured using a single drop spectrophotometer.
I am extracting RNA from fish scales. I took one scale per sample. But the RNA yield was too low and it's homogenization was also difficult. Please help. What can I do to get results?
The RNA concentration will be very low in developed scales. You may have to modify the experimental design. Depends on what your objectives are. Quick suggestion: try flash freezing in liquid Nitrogen.
what extra to follow when using extraction from developing endosperms
This depends on the nature of the endosperm. If liquid as in the case of coconut, you will have to reduce the volume to obtain higher yield using a non destructive method as far as RNA is concerned. If solid, there should be no problem. Most endosperm tissue is triploid (3n), this must be considered when analyzing RNA seq data from endorsperm transcriptomes.
@@KENNETHFRANCISRODRIGUES thanks a lot doc. Actually I need to do RNA extraction of maize at 5, 10 and 15 DAP, so I think on 5th and 10th day, it might be milky.
What other changes I can make.
Thanks a lot for reply.
During these steps there is no need to worry about external RNase contamination? I've seen people do this in RNase free sterile hoods
Thank you for your query. The procedure described above is suitable for the extraction of RNA for downstream application to reverse transcription and reverse transcription quantitative PCR (RT-qPCR), however, it is recommended that a sterile fume hood and the use of appropriate RNase inactivating agents (DEPC) be resorted to in case the RNA will be subjected to RNA sequencing (RNAseq) or in the case of gene transcripts which may be present at a very low copy number in the biological sample.
Thank You! I will be making cDNA so this is good to know.
All the best!
Suppose we want to isolate rna from animal cells? Is the procedure same as u did for plant cell??
Thank you for your query. You will have to use Proteinase K (RNase free) during the lysis step. Commercial kits include this in their RNA extraction procedure.
Hi, can I use this kit and method after RNA extraction using Trizol method as a clean up method?
Thank you. Not advisable as the components from Trizol may interfere with the membrane and interact with other solutions.
It can use for RNA clean-up purpose. After Phase-separation using chloroform, take off the aqueous phase and mix with ethanol, subsequent step is same the this method.
Sir, which kit is recommended to isolate RNA from blood samples?
I used the RNeasy Blood and Tissue Kit. Please ensure DNA cleanup using DNase as there may be carryover of DNA.
@@KENNETHFRANCISRODRIGUES okay. Thanks for the info
@@KENNETHFRANCISRODRIGUES can u plz send the link of that kit for reference
When I follow the provided protocol and this video, RNA was always contaminated by gDNA . I did all steps on the ice, I changed kit, centrifuge machines and all solutions (EtOH). But still have gDNA on RAN tube. How can I solve this problem....?? And I don't want to treat DNase because of the yield decrease
Dear viewer. Thank you for your comments. Generally, you need to perform a DNA removal procedure using DNase. One of the reasons for DNA carryover is overloading with the initial sample. Begin with a small sample volume. Generally for animal tissue less than 200 microlitre volume of tissue.
Excellent effort,
Nice rna
Thank You for the video.
Most welcome.
most helpful sir....thanks a lot
Welcome. All the best.
Thank you Sir.
Why didn't you used 70%ethanol instead of the 100%ethanol?
I think the protocol said 70%ethanol not 100%ethanol!
Thanks!!!
protocol suggests 96-100% ethanol
10 000 g is how many rpm?????????????
Depends on the model of the centrifuge and rotor. The centrifuge in your laboratory will have a conversion table from RPM to xg
Description should be more clear