RNA isolation problem - (Sep/22/2008 )
Hello,
I am using RNA-STAT 60 to isolate total RNA from 3T3-L1 cells grown and differentiated in 6 well plates. I haven't isolated RNA before and I'm having some difficulty with the protocol that I was hoping someone can help me with. Per the protocol, I:
1. Add 0.8 ml RNA-STAT 60 to my cells after aspirating the media (without washing the cells). Scratch the bottom of the well with my tip. Pipet up and down 10-15 times.
2. Add 0.16 ml chlorodofrm, shake, spin 12000g x 15 min at 4 C
3. Transfer aqueous phase to new tube and mix with 0.4 ml of isopropanol. Spin at 12000g x 10 min at 4 C.
*I am supposed to see a pellet (RNA percipitate) at the bottom of the tube after this step and unfortunately, I don't." The rest of the protocol
4. Wash with 0.8 ml 75% ethanol. Spin at 7500g for 5 minutes. Discard supernatant and dry pellet. Add 30 ul of DEPC water to resuspend pellet.
Anyone know where I'm going wrong or suggestions to troubleshoot. Alternatively, does anyone recommend different kits to isolate total RNA for eventual PCR.
Thanks.
-medstudent1-
QUOTE (medstudent1 @ Sep 23 2008, 04:15 AM)
Hello,
I am using RNA-STAT 60 to isolate total RNA from 3T3-L1 cells grown and differentiated in 6 well plates. I haven't isolated RNA before and I'm having some difficulty with the protocol that I was hoping someone can help me with. Per the protocol, I:
1. Add 0.8 ml RNA-STAT 60 to my cells after aspirating the media (without washing the cells). Scratch the bottom of the well with my tip. Pipet up and down 10-15 times.
2. Add 0.16 ml chlorodofrm, shake, spin 12000g x 15 min at 4 C
3. Transfer aqueous phase to new tube and mix with 0.4 ml of isopropanol. Spin at 12000g x 10 min at 4 C.
*I am supposed to see a pellet (RNA percipitate) at the bottom of the tube after this step and unfortunately, I don't." The rest of the protocol
4. Wash with 0.8 ml 75% ethanol. Spin at 7500g for 5 minutes. Discard supernatant and dry pellet. Add 30 ul of DEPC water to resuspend pellet.
Anyone know where I'm going wrong or suggestions to troubleshoot. Alternatively, does anyone recommend different kits to isolate total RNA for eventual PCR.
Thanks.
I am using RNA-STAT 60 to isolate total RNA from 3T3-L1 cells grown and differentiated in 6 well plates. I haven't isolated RNA before and I'm having some difficulty with the protocol that I was hoping someone can help me with. Per the protocol, I:
1. Add 0.8 ml RNA-STAT 60 to my cells after aspirating the media (without washing the cells). Scratch the bottom of the well with my tip. Pipet up and down 10-15 times.
2. Add 0.16 ml chlorodofrm, shake, spin 12000g x 15 min at 4 C
3. Transfer aqueous phase to new tube and mix with 0.4 ml of isopropanol. Spin at 12000g x 10 min at 4 C.
*I am supposed to see a pellet (RNA percipitate) at the bottom of the tube after this step and unfortunately, I don't." The rest of the protocol
4. Wash with 0.8 ml 75% ethanol. Spin at 7500g for 5 minutes. Discard supernatant and dry pellet. Add 30 ul of DEPC water to resuspend pellet.
Anyone know where I'm going wrong or suggestions to troubleshoot. Alternatively, does anyone recommend different kits to isolate total RNA for eventual PCR.
Thanks.
1. so hows ur RNA concentration? is it too low?
2. im using TRIzol reagent (invitrogen) for my total RNA extraction. the main step is similar. i also didnt see the pellet for once ( normally can get to c white pellet after RNA precipitation step ), its because i started with too low amount of cells ( i didnt precipitate my cells before store in -70C ).
3. do u break ur cell before adding the reagent? normally the first step in RNA extraction is breaking the cell ( for my case, i break my cell in liquid nitrogen by using mortar and pestle-traditional way ). the adding of chloroform will separate the sample into two layer, upper aquous layer with RNA while lower layer containing DNA.
so, the cell have to be break down completely, in order to release all the rna from the cell...
jus a bit sharing from wat i know.. hope will be help..
-parami-
As parami said maybe the cells didn't lysed. Can use a syringe with a very fine needle (25g) the one use for tuberculin is good. The tips aren't fine enough so the cell can remain intact.
-merlav-
QUOTE (medstudent1 @ Sep 22 2008, 01:15 PM)
I am using RNA-STAT 60 to isolate total RNA from 3T3-L1 cells grown and differentiated in 6 well plates. I haven't isolated RNA before and I'm having some difficulty with the protocol that I was hoping someone can help me with.
*I am supposed to see a pellet (RNA percipitate) at the bottom of the tube after this step and unfortunately, I don't." The rest of the protocol
*I am supposed to see a pellet (RNA percipitate) at the bottom of the tube after this step and unfortunately, I don't." The rest of the protocol
I usually don't see a pellet at the isoproponal stage unless i have a ton of cells... more than 3 million cells or so. In my experience, I found the RNA-stat 60 to yield lower quality RNA at a lower quantity as well. Invitrogen's trizol is a better product IMO. Also when washing with ethanol make sure it is very cold. You can have it at -80C and it will give you a better yield than ethanol at room temp.
-rubyeye-
Hi,
I am also having problems with miniprep RNA preparation using T7 Kit.
I dont kno where did i go wrong that i get plasmids from E.coli, and i able to get linearized plasmids but when i make RNA, i dont get that.
cAN anyone suggest something???
-SYDNEY-
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
-Trof-
QUOTE (Trof @ Oct 20 2008, 08:51 AM)
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
hi trof and all,
thanks for all of the advice. good pointer about putting the tube in the same direction each time. as an update, ive been using RNA-STAT 60 and things have been working decently. ive been able to extract rna (approximately 1 ug/ml) and subsequently make cDNA for qPCR. i am isolating rna from 6 well plates so you are right about the pellet - i have been going on "good faith" when pouring off my supernatant because as you mention i dont see a pellet.
-medstudent1-
QUOTE (medstudent1 @ Oct 21 2008, 09:07 PM)
QUOTE (Trof @ Oct 20 2008, 08:51 AM)
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
hi trof and all,
thanks for all of the advice. good pointer about putting the tube in the same direction each time. as an update, ive been using RNA-STAT 60 and things have been working decently. ive been able to extract rna (approximately 1 ug/ml) and subsequently make cDNA for qPCR. i am isolating rna from 6 well plates so you are right about the pellet - i have been going on "good faith" when pouring off my supernatant because as you mention i dont see a pellet.
Hi All,
Yes i use ice cold ethanol but 70% with DEPC. i do put the tubes in the same direction so i know which side most probably would because you know
literally the pellet is invisible. I want to ask when i drain teh liquid i usually do that with pipett , means i remove the supernatant with pipett, i sthat ok or just i should simply drained it from eppendorf?
sydney
-SYDNEY-
QUOTE (SYDNEY @ Oct 22 2008, 03:01 PM)
QUOTE (medstudent1 @ Oct 21 2008, 09:07 PM)
QUOTE (Trof @ Oct 20 2008, 08:51 AM)
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
hi trof and all,
thanks for all of the advice. good pointer about putting the tube in the same direction each time. as an update, ive been using RNA-STAT 60 and things have been working decently. ive been able to extract rna (approximately 1 ug/ml) and subsequently make cDNA for qPCR. i am isolating rna from 6 well plates so you are right about the pellet - i have been going on "good faith" when pouring off my supernatant because as you mention i dont see a pellet.
Hi All,
Yes i use ice cold ethanol but 70% with DEPC. i do put the tubes in the same direction so i know which side most probably would because you know
literally the pellet is invisible. I want to ask when i drain teh liquid i usually do that with pipett , means i remove the supernatant with pipett, i sthat ok or just i should simply drained it from eppendorf?
sydney
Hi,
I have a similar problem but the difference is that I saw a white pellet when I washed my cells with 75% Etoc (made in DEPC water). I air dried it for 20mins and the white pellet went invisible (see through in colour). Anothertube I was working on has teh white coloured pellet after air drying it for the same amount f time. To play safe, I dissolved both pellets in DEPC treated water and incuated in 60 deg water bath. I have not checked the concentration of both the RNA's but any idea why one pellet went from white to clear? Did I over dry my pellet?
-Sarwat-
QUOTE (Sarwat @ Oct 23 2008, 02:46 AM)
QUOTE (SYDNEY @ Oct 22 2008, 03:01 PM)
QUOTE (medstudent1 @ Oct 21 2008, 09:07 PM)
QUOTE (Trof @ Oct 20 2008, 08:51 AM)
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
hi trof and all,
thanks for all of the advice. good pointer about putting the tube in the same direction each time. as an update, ive been using RNA-STAT 60 and things have been working decently. ive been able to extract rna (approximately 1 ug/ml) and subsequently make cDNA for qPCR. i am isolating rna from 6 well plates so you are right about the pellet - i have been going on "good faith" when pouring off my supernatant because as you mention i dont see a pellet.
Hi All,
Yes i use ice cold ethanol but 70% with DEPC. i do put the tubes in the same direction so i know which side most probably would because you know
literally the pellet is invisible. I want to ask when i drain teh liquid i usually do that with pipett , means i remove the supernatant with pipett, i sthat ok or just i should simply drained it from eppendorf?
sydney
Hi,
I have a similar problem but the difference is that I saw a white pellet when I washed my cells with 75% Etoc (made in DEPC water). I air dried it for 20mins and the white pellet went invisible (see through in colour). Anothertube I was working on has teh white coloured pellet after air drying it for the same amount f time. To play safe, I dissolved both pellets in DEPC treated water and incuated in 60 deg water bath. I have not checked the concentration of both the RNA's but any idea why one pellet went from white to clear? Did I over dry my pellet?
I think you should dry it for just 5 minutes not 20 minutes.
-SYDNEY-