Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis
Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).
1. A method for determination of stable isotope incorporation in a community of organisms comprising the steps of:
a) supplying said community of organisms with at least two substrates simultaneously for a defined period of time, wherein each of the at least two substrates is labeled with a different stable isotope;
b) extracting RNA from the organisms;
c) fragmenting said RNA to provide fragmented RNA;
d) labeling a fraction of the fragmented RNA with a detectable label to provide labeled fragmented RNA;
e) hybridizing the labeled fragmented RNA to a set of oligonucleotide probes, wherein the set of oligonucleotide probes is an array of oligonucleotide probes attached to a substrate;
f) detecting hybridization signal strength of labeled fragmented RNA hybridized to the oligonucleotide probes to determine the community organism composition;
g) identifying a responsive set of oligonucleotide probes based on the hybridization signal strength in step f);
h) hybridizing a fraction of unlabeled fragmented RNA to a second array of oligonucleotide probes, wherein the second array comprises the responsive set of oligonucleotide probes attached to a conductive substrate;
i) detecting the unlabeled fragmented RNA hybridized to the responsive set of probes to determine the stable isotope incorporation into the community of organisms using imaging mass spectrometry or spectroscopy.
2. The method of claim 1 , wherein said organism is a bacterium, archaea, virus, fungus, plant, arthropod, nematode, or other eukaryote.
3. The method of claim 2 , wherein said organism is a bacterium.
4. The method of claim 1 , wherein the stable isotopes are selected from the group consisting of 3 H, 13 C, 15 N, and 18 O.
5. The method of claim 1 , wherein in step b) the extracting step is carried out by physical or chemical cell lysis followed by affinity column purification.
6. The method of claim 1 , wherein in step c) the fragmenting step is carried out by using enzymes, chemicals, or heat, or a combination of these.
7. The method of claim 1 , wherein in step d) the RNA is labeled with a fluorescent molecule or a non-fluorescent molecule.
8. The method of claim 1 , wherein steps c) and d) are carried out concurrently.
9. The method of claim 1 , wherein step e) further comprises the steps of adding said labeled fragmented RNA to a hybridization solution and contacting said hybridization solution with the array of oligonucleotide probes.
10. The method of claim 1 , wherein the set of oligonucleotide probes comprises 16S rRNA phylogenetic oligonucleotide probes.
11. The method of claim 10 , wherein said set of 16S rRNA phylogenetic probes further comprises probes from the 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, cox1 gene, nif13 gene, or a combination thereof.
12. The method of claim 7 , wherein the RNA is labeled with a fluorescent molecule.
13. The method of claim 7 , wherein the RNA is labeled with a non-fluorescent molecule.
14. The method of claim 1 , wherein in step f) the hybridized labeled RNA is imaged with a fluorescence scanner and fluorescence intensity is measured for each probe feature.
15. The method of claim 1 , wherein in step f) the detection of hybridization signal strength provides a determination of genes present in the community of organisms.
16. The method of claim 1 , wherein in step f) the detection of hybridization signal strength is used for normalization of the isotope signals detected in step i).
17. The method of claim 1 , wherein in step i) the hybridized unlabeled fragmented RNA are imaged with a secondary ion mass spectrometer and isotope ratios are measured for each probe feature.
18. The method of claim 1 , wherein in step i) the hybridized unlabeled fragmented RNA are imaged with a nano-scale secondary ion mass spectrometer device and isotope ratios are measured for each probe feature.
19. The method of claim 1 , wherein in step e) the substrate is a solid planar substrate, a microarray slide, spheres or beads, wherein the substrate is comprised of silicon, glass, metals, semiconductor materials, polymers or plastics.
20. The method of claim 18 , wherein in step h) the responsive probe substrate is a solid planar substrate, a microarray slide, spheres or beads, wherein the responsive probe substrate is comprised of silicon, glass, metals, semiconductor materials, polymers or plastics.
21. The method of claim 20 , wherein the responsive probe substrate is a solid planar substrate coated with indium tin oxide (ITO).
22. The method of claim 1 , wherein in step e) the set of oligonucleotide probes is identified and selected from unique sequence regions of the fragmented RNA.
23. A method for determination of stable isotope incorporation in a community of organisms comprising the steps of:
a) supplying said community of organisms with at least two substrates simultaneously for a defined period of time, wherein each of the at least two substrates is labeled with a different stable isotope;
b) extracting RNA from the organisms;
c) fragmenting said RNA to provide fragmented RNA, generating cDNAs from a fraction of said fragmented RNA and sequencing said cDNAs;
d) designing a first set of oligonucleotide probes based on unique sequence regions of said sequenced cDNAs generated from the fragmented RNA;
e) labeling a fraction of the fragmented RNA with a detectable label to provide labeled fragmented RNA;
f) hybridizing the labeled fragmented RNA to the first set of oligonucleotide probes, wherein the first set of oligonucleotide probes is an array of oligonucleotide probes attached to a substrate, and detecting hybridization signal strength of the labeled fragmented RNA hybridized to the first set of oligonucleotide probes to determine the community organism composition;
g) identifying a responsive set of oligonucleotide probes based on the hybridization signal strength in step f);
h) hybridizing a fraction of unlabeled fragmented RNA to a second array of oligonucleotide probes, wherein the second array comprises the responsive set of oligonucleotide probes attached to a conductive substrate;
i) detecting the unlabeled fragmented RNA hybridized to the responsive set of probes to determine the stable isotope incorporation into the community of organisms using imaging mass spectrometry or spectroscopy.
24. The method of claim 23 , wherein said organism is a bacterium, archaea, virus, fungus, plant, arthropod, nematode, or other eukaryote.
25. The method of claim 24 , wherein said organism is a bacterium.
26. The method of claim 23 , wherein the stable isotopes are selected from the group consisting of 3 H, 13 C, 15 N, and 18 O.
27. The method of claim 23 , wherein in step b) the extracting step is carried out by physical or chemical cell lysis followed by affinity column purification.
28. The method of claim 23 , wherein in step c) the fragmenting step is carried out by using enzymes, chemicals, or heat, or a combination of these.
29. The method of claim 23 , wherein in step e) the RNA is labeled with a fluorescent molecule or a non-fluorescent molecule.
30. The method of claim 23 , wherein steps c) and e) are carried out concurrently.
31. The method of claim 23 , wherein step f) further comprises the steps of adding said labeled fragmented RNA to a hybridization solution and contacting said hybridization solution with the array of oligonucleotide probes.
32. The method of claim 23 , wherein the set of oligonucleotide probes comprises 16S rRNA phylogenetic oligonucleotide probes.
33. The method of claim 32 , wherein said set of 16S rRNA phylogenetic probes further comprises probes from the 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, cox1 gene, nif13 gene, or a combination thereof.
34. The method of claim 29 , wherein the RNA is labeled with a fluorescent molecule.
35. The method of claim 29 , wherein the RNA is labeled with a non-fluorescent molecule.
36. The method of claim 23 , wherein in step f) the hybridized labeled RNA is imaged with a fluorescence scanner and fluorescence intensity is measured for each probe feature.
37. The method of claim 23 , wherein in step f) the detection of hybridization signal strength provides a determination of genes present in the community of organisms.
38. The method of claim 23 , wherein in step f) the detection of hybridization signal strength is used for normalization of isotope signals detected in step i).
39. The method of claim 23 , wherein in step i) the hybridized unlabeled fragmented RNA are imaged with a secondary ion mass spectrometer and isotope ratios are measured for each probe feature.
40. The method of claim 23 , wherein in step i) the hybridized unlabeled fragmented RNA are imaged with a nano-scale secondary ion mass spectrometer device and isotope ratios are measured for each probe feature.
41. The method of claim 23 , wherein in step f) the substrate is a solid planar substrate, a microarray slide, spheres or beads, wherein the substrate is comprised of silicon, glass, metals, semiconductor materials, polymers or plastics.
42. The method of claim 23 , wherein in step h) the responsive probe substrate is a solid planar substrate, a microarray slide, spheres or beads, wherein the responsive probe substrate is comprised of silicon, glass, metals, semiconductor materials, polymers or plastics.
43. The method of claim 25 , wherein the responsive probe substrate is a solid planar substrate coated with indium tin oxide (ITO).