Compositions and methods for internal controls of microvesicle isolations
The present disclosure provides compositions, methods and kits for internal controls of microvesicle isolations. The compositions, methods and kits can comprise enveloped viruses, including, but not limited to, inactive mouse hepatitis virus (MHV).
1. A method comprising:
(a) adding a known quantity of control particles comprising at least one control nucleic acid and/or at least one control protein to a biological sample, wherein the control particles comprise a non-human animal enveloped virus, wherein the non-human animal enveloped virus is selected from mouse hepatitis virus (MHV), bovine diarrhea virus (BVDV) and transmissible gastroenteritis coronavirus (TGEV);
(b) isolating microvesicles and the control particles from the biological sample;
(c) extracting nucleic acids and/or protein from the microvesicles and the control particles;
(d) assaying the amount of the at least one control nucleic acid and/or the at least one control protein recovered, thereby determining the amount of control particles recovered;
(e) determining that the amount of control particles calculated in step (d) is within a predetermined range of values; and
(f) subjecting the nucleic acids and/or protein from step (c) to further analysis if the amount of control particle calculated in step (d) is within the predetermine range of values;
wherein the isolating of the microvesicles and control particles from the biological sample comprises contacting the biological sample with a capture surface under conditions sufficient to retain cell-free DNA, microvesicles, and control particles on the capture surface, and
wherein the capture surface comprises one or more beads that are positively charged, one or more beads that are an anion exchanger functionalized with quaternary ammonium (R—CH 2 —N + (CH 3 ) 3 ), or one or more beads that are positively charged and functionalized with quaternary ammonium.
2. The method of claim 1 , wherein the non-human animal enveloped virus is an inactivated non-human animal enveloped virus.
3. The method of claim 1 , wherein the biological sample is a bodily fluid sample, wherein the bodily fluid sample comprises urine, blood, cerebrospinal fluid, serum or any combination thereof.
4. The method of claim 1 , wherein the at least one control nucleic acid comprises RNA.
5. The method of claim 1 , wherein the nucleic acids from step (c) comprises RNA, DNA, or RNA and DNA.
6. The method of claim 1 , wherein assaying the amount of the at least one control nucleic acid recovered comprises determining the expression level or copy number of the control nucleic acid.
7. The method of claim 6 , wherein assaying the expression level or copy number of the control nucleic acid comprises reverse transcribing the control nucleic acid, quantitative PCR (qPCR), or any combination thereof.
8. The method of claim 1 , wherein step (f) further comprises determining the presence, absence or level of at least one biomarker associated with a disease or medical condition for diagnosing, prognosing, or monitoring the disease or medical condition.
9. The method of claim 8 , wherein determining the presence, absence or level of at least one biomarker associated with a disease or medical condition for diagnosing, prognosing, or monitoring the disease or medical condition comprises reverse transcription, quantitative PCR, or any combination thereof.
10. A method comprising:
(a) isolating microvesicles from a biological sample;
(b) adding a known quantity of control particles comprising at least one control nucleic acid and/or at least one control protein to the isolated microvesicles, wherein the control particles comprise a non-human animal enveloped virus, wherein the non-human animal enveloped virus is selected from mouse hepatitis virus (MHV), bovine diarrhea virus (BVDV) and transmissible gastroenteritis coronavirus (TGEV);
(c) extracting nucleic acids and/or protein from the isolated microvesicles and the control particles;
(d) assaying the amount of the at least one control nucleic acid and/or the at least one control protein recovered, thereby determining the amount of control particles recovered;
(e) determining that the amount of control particles calculated in step (d) is within a predetermined range of values; and
(f) subjecting the nucleic acids and/or protein from step (c) to further analysis if the amount of control particle calculated in step (d) is within the predetermine range of values;
wherein the isolating of the microvesicles and control particles from the biological sample comprises contacting the biological sample with a capture surface under conditions sufficient to retain cell-free DNA, microvesicles, and control particles on the capture surface, and
wherein the capture surface comprises one or more beads that are positively charged, one or more beads that are an anion exchanger functionalized with quaternary ammonium (R—CH 2 —N + (CH 3 ) 3 ), or one or more beads that are positively charged and functionalized with quaternary ammonium.
11. The method of claim 10 , wherein the non-human animal enveloped virus is an inactivated non-human animal enveloped virus.
12. The method of claim 10 , wherein the biological sample is a bodily fluid sample, wherein the bodily fluid sample comprises urine, blood, cerebrospinal fluid, serum or any combination thereof.
13. The method of claim 10 , wherein the at least one control nucleic acid comprises RNA.
14. The method of claim 10 , wherein the nucleic acids from step (c) comprises RNA, DNA, or RNA and DNA.
15. The method of claim 10 , wherein assaying the amount of the at least one control nucleic acid recovered comprises assaying the expression level or copy number of the control nucleic acid.
16. The method of claim 15 , wherein assaying the expression level or copy number of the control nucleic acid comprises reverse transcribing the control nucleic acid, quantitative PCR (qPCR), or any combination thereof.
17. The method of claim 10 , wherein step (f) further comprises determining the presence, absence or level of at least one biomarker associated with a disease or medical condition for diagnosing, prognosing, or monitoring the disease or medical condition.
18. The method of claim 7 , wherein the quantitative PCR is reverse transcription quantitative PCR (RT-qPCR).
19. The method of claim 16 , wherein the quantitative PCR is reverse transcription quantitative PCR (RT-qPCR).